Tissue Culture Inserts for Multiwell Plates - file.yizimg.comfile.yizimg.com/472786/2013071112040270.pdf · Greiner Bio-One offers tissue culture inserts for 6, 12 und 24 well multiwell

www.gbo.com/bioscience Germany: Greiner Bio-One GmbH, [email protected] l Austria: Greiner Bio-One GmbH, [email protected] Belgium: Greiner Bio-One BVBA/SPRL, [email protected] l Brazil: Greiner Bio-One Brasil, [email protected] China: Greiner Bio-One GmbH, [email protected] l France: Greiner Bio-One SAS, [email protected] Japan: Greiner Bio-One Co. Ltd., [email protected] l Netherlands: Greiner Bio-One B.V., [email protected] UK: Greiner Bio-One Ltd., [email protected] l USA: Greiner Bio-One North America Inc., [email protected]

Revision: October 2003 – F075001

ThinCert TM Tissue Culture Inserts for Multiwell Plates

Greiner Bio-One offers tissue culture inserts for 6, 12 und 24 well multiwell plates with 0.4 µm, 1.0 µm, 3.0 µm and 8.0 µm pore sizes.

F e a t u r e s

� stable housing made of highly transparent polystyrene

� hanging geometry

� sealed PET capillary pore membrane

� improved cell adhesion due to physical surface treatment

� single, sterile blister packing

� simplified pipetting due to self lift geometry

� notches at the upper edge allow optimized gas exchange

O r d e r i n g I n f o r m a t i o n ThinCert TM and Multiwell Plate

Size

Membranematerial

Culture Surface [mm 2]

Pore Size [µm]

Pore Density [cm -2]

Optical Membrane Properties

ThinCert TM / Plates per

Box Cat.-No.

6 well PET 452,4 0.4 1 x 108 translucent 24 / 4 657 640

6 well PET 452,4 0.4 2 x 106 transparent 24 / 4 657 641

6 well PET 452,4 1.0 2 x 106 transparent 24 / 4 657 610

6 well PET 452,4 3.0 0.6 x 106 transparent 24 / 4 657 630

6 well PET 452,4 3.0 2 x 106 translucent 24 / 4 657 631

6 well PET 452,4 8.0 0.15 x 106 translucent 24 / 4 657 638

12 well PET 113.1 0.4 1 x 108 translucent 48 / 4 665 640

12 well PET 113.1 0.4 2 x 106 transparent 48 / 4 665 641


12 well PET 113.1 3.0 0.6 x 106 transparent 48 / 4 665 630


12 well PET 113.1 8.0 0.15 x 106 translucent 48 / 4 665 638




24 well PET 33.6 3.0 0.6 x 106 transparent 48 / 2 662 630


24 well PET 33.6 8.0 0.15 x 106 translucent 48 / 2 662 638

www.gbo.com/bioscience

Application NoteCo-culture in ThinCert™ cell culture inserts

Item Manufacturer Cat.-No.

Alexa Fluor 488 anti rabbitIgG antibody

Invitrogen GmbH A11008

Beta estradiol Sigma-Aldrich Chemie GmbH E8875

Cell proliferation kit I (MTT) Roche Diagnostics 1465007

CELLSTAR® 24 well cell culture plate

Greiner Bio-One GmbH 662 160

DakoCytomation FluorescentMounting Medium

Dako Deutschland GmbH S3023

DAPI, dilactate Sigma-Aldrich Chemie GmbH D9564

DMEM medium Biochrom AG F0435

Fetal calf serum Invitrogen Life Technologies 10270-106

Formalin Sigma-Aldrich Chemie GmbH HT5014

Insulin (bovine) Biochrom AG K 3510

Ki67 antibody Abcam Ab15580

L-alanyl-L-glutamine Biochrom AG K0302

PBS-Dulbeco Biochrom AG L1825

RPMI medium Biochrom AG F1295

ThinCert™ 24 well cell cul-ture insert with 0.4 μm pores


Tritron X100 Sigma-Adrich Chemie GmbH T8787

Figure 1 Different modes of co-culture using ThinCert™ cell culture inserts.Two cell populations that are co-cultivated in different compartments (insert and well) stay physically separated, but may communicate via paracrine signallingthrough the pores of the membrane (A, B). Alternatively, both cell populations may be co-cultivated in the upper compartment (insert), thus allowing extensive and direct cell-cell interactions (C).

Material

2 4 www.gbo.com/bioscience

Introduction

Co-culture describes various techniques where different cellpopulations are cultivated in close proximity in the same cellculture environment. The applications of co-cultures are multi-facetted and include:

� stimulation and maintenance of cell function and differentiation,

� cultivation of embryonic stem cells on feeder cells,� applications in reproductive medicine (e.g. autologous

endometrial co-culture),� investigation of immune cell interactions,� investigation of paracrine mesenchymal-epithelial

interactions,� restoration of heterocellular functions in vitro

(e.g. blood-brain-barrier).

Direct co-culture can be performed in nearly all cell culturedishes, for instance by layering two cell types one on top ofthe other. In contrast, indirect co-culture takes advantage ofcell culture inserts with porous membranes, to keep the co-cultivated cell populations separated (Fig. 1).

Co-cultures with cell culture inserts have been widely used tostudy mesenchymal-epithelial interactions during normal andtumoral development (Hofland et al., 1995; Gache et al.,1998). Here, such a co-culture model has been establishedusing MCF7 mammary carcinoma cells, human fibroblastsand ThinCert™ cell culture inserts. These experiments illus-trate the excellent suitability of ThinCert™ cell culture insertsfor co-culture applications.

The following protocol presents technical details for co-cultureand may be easily adapted to match individual requirementsand research interests other than paracrine growth regulation.

Cell layer

Interactive Cell layer Porous PET membrane Interactive cell layer inextracellular matrix

ThinCertTM

cell culture insert

Well of multiwellplate

Medium

(A) (B) (C)

Methods

Seeding of fibroblasts onto the underside of the membrane and co-culture

24 well ThinCert™ cell culture inserts with translucent membranes and 0.4 μm pores were inverted and placed in a 12 well plate (Fig. 2/1). The well bottom was humidified with100 μl sterile water (Fig. 2/2). 60 μl of a cell suspension containing 0 (control); 83,000; 167,000 or 418,000 human juvenile foreskin fibroblasts per ml DMEM medium (supplemented with 10% FCS, 4 mM L-alanyl-glutamine) waspipetted into the inner circle of the underside of the membrane(Fig. 2/3, 2/4).

The plate was covered with a lid, thus holding the cell suspension to the underside of the membrane by capillaryforces (Fig. 2/5). The cells were allowed to adhere overnight at37°C and 5% CO2. Subsequently, the insert was placed in thewell of a 24 well plate pre-filled with 800 μl RPMI medium containing 10 μg/ml insulin, 110 pM estradiol, 10% FCS and 4 mM L-alanyl-glutamine (Fig. 2/6). 200 μl MCF7 suspensioncontaining 25,000 cells per ml supplemented RPMI mediumwas added to each insert. Co-cultures were maintained for 2days at 37°C and 5% CO2.

___________1 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide


MTT1 assay

The MTT assay was performed with the Cell Proliferation Kit Ifrom Roche. After removal of the cell culture medium from theinserts, the fibroblasts were wiped off the underside of the insert using a cotton swab. The inserts were placed in afreshly prepared 24 well plate containing 400 μl MTT medium (0.5 mg/mL) per well.

100 μl MTT medium was added to each insert. After an incubation of 4 h at 37 °C and 5% CO2, 400 and 100 μl solubilization solution was added to each well and insert, respectively. The next day, 200 μl of the combined and mixedsolutions from the insert and well were transferred to a clearbottom 96 well plate. The absorbance was measured with aTECAN Safire plate reader at 570 nm.

Results and discussion

Mesenchymal-epithelial interactions have been shown to playan important role in normal breast development and breasttumorigenesis. In vivo, positive feedback loops between hormone responsive breast tumor cells and their surroundingfibroblasts seem to account for enhanced tumor growth andare likely mediated by hormones and growth factors exchanged between both cell populations (reviewed in Clarkeet al., 1992). Previously, indirect and direct co-cultures ofbreast tumor cells and fibroblasts have been extensively usedto study mesenchymal-epithelial interactions in breast tumorformation in vitro (Hofland et al., 1995; Gache et al., 1998; Heneweer et al., 2005). Indirect co-cultures using cell cultureinserts revealed the paracrine growth promoting effect ex-erted by fibroblasts on breast tumor cells (Hofland et al.,1995; Gache et al., 1998).

Here, such a co-culture model has been established usingMCF7 breast cancer cells and human fibroblasts that wereco-cultivated on the upper and lower sides of the membraneof ThinCert™ cell culture inserts, respectively. ProliferativeMCF7 cells could be identified on the basis of positive Ki67immunoreactivity (Fig. 3).

Figure 2Cell seeding onto the underside of the insert membrane. A 24 well insert isplaced upside down into a humidified well of a 12 well plate (1, 2). Cell sus-pension is pipetted onto the membrane underside (3, 4). The plate is closedwith a lid (5). After overnight incubation (37ºC, 5% CO2) the insert is placed ina 24 well plate. A second cell population may now be seeded into the uppercompartment (6).

Figure 3Identification of non-proliferative cells (arrowheads) based on lack of Ki67 immunoreactivity.

Quantification of proliferative cells by Ki67 immunocyto-chemistry

The immunocytochemistry protocol used here is described in our corresponding application note (No. 073 100)(www.gbo.com/bioscience/thincert). In brief, cells in the insertwere fixed with 500 μl 4% formalin, washed twice with 500 μlPBS and permeabilised for 25 min with 500 μl 0.5% Triton/PBS. After washing with PBS, non-specific protein bindingsites were blocked with 500 μl 10% FCS/PBS for 1.5 h.

Cells were washed with PBS and incubated for 1 h with 100 μlrabbit anti Ki67 antibody (1:100 in 1% FCS/PBS). After wash-es with PBS, the cells were incubated for 1 h with 100 μlAlexa 488 anti rabbit IgG antibody (1:250 in 1% FCS/PBS).Nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole). Insert membranes were cut out and mountedon microscopy slides using fluorescence mounting medium.

For analysis, the number of Ki67 positive cells and the totalcell number (DAPI) per microscopic field were counted usingan inverted microscope at a 250x magnification. A two-tailedt-test was used for comparison between two experimentaldata sets. A value of P<0.05 was considered statistically significant.


Germany (Main office)Greiner Bio-One GmbHPhone: (+49) 7022 948-0E-Mail: [email protected]

AustriaGreiner Bio-One GmbHPhone: (+43) 7583 6791-0E-Mail: [email protected]

BelgiumGreiner Bio-One N. V.Phone: (+32) 2-4 61 09 10E-Mail: [email protected]

NetherlandsGreiner Bio-One B. V.Phone: (+31) 172-42 09 00E-Mail: [email protected]

FranceGreiner Bio-One SASPhone: (+33) 169-86 25 50E-Mail: [email protected]

UKGreiner Bio-One Ltd.Phone: (+44) 1453-82 52 55E-Mail: [email protected]

JapanGreiner Bio-One Co. Ltd.Phone: (+81) 3-35 05-88 75E-Mail: [email protected]

USAGreiner Bio-One North America Inc.Phone: (+1) 704-261-78 00E-Mail: [email protected]

Revision: November 2007- 074 059

Human fibroblasts demonstrated a clear growth promoting effect on MCF7 cells. This effect increased in a dose dependent manner with the number of applied fibroblasts. It was reflected in:(1) an increased number of Ki67 positive MCF7 cells

(Fig. 4/A),(2) an increased total number of MCF7 cells (Fig. 4/B),(3) an elevated ratio of Ki67 positive cells vs. total cell number

(Fig. 4/C), and(4) an increased MTT metabolism of the MCF7 cell population

(Fig. 5).

For instance, as compared to single culture conditions, theco-cultivation of MCF7 cells with 25,000 fibroblasts yielded a2.2-fold higher final cell number and a 1.2-fold larger fractionof proliferative cells (Fig. 4).

References

Clarke R, Dickson RB, Lippman ME. (1992) Hormonal aspects of breastcancer. Growth factors, drugs and stromal interactions. Crit Rev Oncol Hematol. Jan;12(1):1-23.

Gache C, Berthois Y, Martin PM, Saez S. (1998) Positive regulation of normal and tumoral mammary epithelial cell proliferation by fibroblasts incoculture. In Vitro Cell Dev Biol Anim. Apr;34(4):347-51.

Heneweer M, Muusse M, Dingemans M, de Jong PC, van den Berg M,Sanderson JT. (2005) Co-culture of primary human mammary fibroblastsand MCF-7 cells as an in vitro breast cancer model. Toxicol Sci.Feb;83(2):257-63.

Hofland LJ, van der Burg B, van Eijck CH, Sprij DM, van Koetsveld PM,Lamberts SW. (1995) Role of tumor-derived fibroblasts in the growth of primary cultures of human breast-cancer cells: effects of epidermal growthfactor and the somatostatin analogue octreotide. Int J Cancer. Jan3;60(1):93-9.

Figure 4Growth promoting effect of primary human fibroblasts on MCF7 cells. Fibroblasts cultivated on the underside of ThinCert™ cell culture inserts exert a growth promoting effect on MCF7 cells cultivated on the upper side of the membrane. The effect manifests in (A) the number of proliferating (Ki67 positive) MCF7 cells, (B) the total number of MCF7 cells and (C) the ratio between proliferative vs. total cell numbers. Averages and standard errors are shown. Statistically significant differences are marked with asterisks (P<0.05).

Figure 5MTT metabolism of MCF7 cells under single cultureand co-culture conditions. Averages and standarderrors are shown.

All parameters were assessed after two days in co-culturewith identical original seeding densities of 5,000 MCF7 cellsper insert.

The co-culture model established here proved to be a helpfultool for studying the paracrine interaction of different cell populations in vitro. The use of small pores (0.4 μm) guarantees that primarily paracrine signaling and not directcell-cell contact accounts for the observed effects. Many variations of this model are conceivable, such as the application of larger pores (3.0 μm) that may allow direct cell-cell contact and therefore additional interaction to occur between the co-cultivated cell populations.

Ki6

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Human fibroblasts per insert


MTT

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F7 c

ells

[OD

570]


Ki6

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(A) (B) (C)


Application NoteTissue reconstruction using ThinCert™ Cell Culture Inserts and Plates


Accu-Chek® Aviva BloodGlucose Meter

Roche 3360578

Accu-Chek® Aviva strips forGlucose Meter

Roche 3360561

Bouin's fluid Sigma-Aldrich Chemie GmbH HT101128

CELLSTAR®, 12 well cell culture plate


Chondroitin 4-sulfate Fluka 27042

Chondroitin 6-sulfate Fluka 27043

DMEM, liquid medium Invitrogen 41965-039

DMEM, powder Invitrogen 52100-021

EnVision™+ System-HRP(AEC)

Dako K4004

Fetal calf serum Invitrogen 10270-106

Fibronectin Invitrogen 33016-015

HEPES-Buffer Sigma-Aldrich Chemie GmbH H4034

KBM Basal Medium Cambrex Bio Science CC-3101

Keratinocyte-SFMMedium (Kit)

Invitrogen 17005-075

KGM SingleQuot Kit Cambrex Bio Science CC-4131

Mouse anti Cytokeratin 10antibody

DakoCytomation M7002

Mouse anti Cytokeratin 5/6antibody

DakoCytomation M7237

Mouse anti Filaggrin antibody

Biomeda V10118

ThinCert™Plate, 12 well tissue culture plate


ThinCert™12 well Cell Culture Insert with 0,4 μmpores


Tissue-Tek® Cryo-OCT Compound

Fisher Scientific 14-373-65

Introduction

For many years live-animal experimentation provided the singular means to access intact and living tissue for biologicaland pharmaceutical research. This situation changed with the introduction of new cell culture approaches that enabled reconstruction of tissue by multi-layering individual cell types in vitro.

Such approaches typically involve the use of porous membranesupports and/or three-dimensional collagen gels to re-create in vivo-like growth within a cell culture environment (Bell et al.,1979; Naughton et al., 1989). With the availability of primarycells from biopsies and organ donations, the spectrum of reconstructed tissues may be expanded to additionally includehuman material. Today, reconstructed renal and intestinal epithelia, epidermis, full thickness skin, airway epithelia, corneaand oral epithelia are frequently used to assess corrosive, irritating and phototoxic potential of substances as well as tostudy drug delivery and pathology.

One of the main challenges in tissue reconstruction is to restorespecific tissue functions in vitro. Depending on the particular tissue type and scientific question, such critical features may encompass barrier function, the capability of substance transport, and the potential to express certain marker genes and signaling molecules. Optimal cell culture conditions must bewell-established to re-create native function and maintain suchfeatures in vitro. For instance, it has been determined thatcultivated epidermal cells differentiate and form a coherentstratum corneum only if they are exposed to air (Asselineau etal., 1985; Ponec et al., 1988). Specific cell culture labware, suchas ThinCert™ Cell Culture Inserts with porous membranesupports, allows cultivation of epidermal cells at the air-liquidinterface (air-lift culture) to ensure formation of a stratumcorneum.

Figure 1 illustrates the two major steps of the formation of askin equivalent in vitro including the submersed cultivation andthe air-lift culture of the human skin model. With ThinCert™ cell culture inserts and the novel ThinCert™Plate Greiner Bio-One offers an integrated solution for multipletissue reconstruction applications. In the following, theopportunities and advantages of ThinCert™ Cell Culture Products are illustrated with two reconstructed tissue models –a multi-layered buccal mucosa and a full thickness skin.

Material


Methods

Cultivation of human oral squamous keratinocytes

ThinCert™ 12 well Cell Culture Inserts with 0.4 μm membraneswere placed in the wells of 12 well standard plates or ThinCert™Plates. 500,000 oral squamous keratinocytes were seeded ineach insert. After overnight incubation at 37°C/ 5% CO2

excessive medium was removed from the upper insert compartment and the cells were further cultivated at the air-liquid-interface using 0.5 and 4 ml Keratinocyte-SFMmedium (with EGF, BPE) per well in the standard plate and ThinCert™Plate, respectively. Medium was exchanged everyday in regular plates and every 4th day in ThinCert™Plates. Cellswere cultivated for 29 days, thus forming a multi-layered buccalmucosa-like epithelium.

Preparation and cultivation of full thickness skin models

For gel solution 232.5 ml 2x DMEM cell culture medium, 7.5 mlHEPES buffer (4.76% in PBS, pH 7.3) and 1.25 ml chondroitinsulfate solution (5 mg/ml chondroitin 4-sulfate, 5 mg/mlchondroitin 6-sulfate in PBS+1) were mixed. The pH was ad-justed to 7.8. Collagen gel (collagen isolated from rat tail ten-don) containing 2 volume shares collagen solution (6 mg/ml in0.1 % acetic acid) and 1 volume share gel solution wasprepared according to patent WO 0192477 A2. 750 μl gelcontaining 75,000 human foreskin fibroblasts was pipetted ontop of the membrane of a 0.4 μm 12 well ThinCert™ CellCulture insert in a standard 12 well cell culture plate. Followinggel formation at 37°C, the gel was covered with 50 μl fibronectinsolution (5 μg/ml) and incubated for 10 min at 37°C. Thefibroblast-containing gel was cultivated for 1 day at 37°C and5% CO2 under submersed conditions applying a total of 2.5 mlDMEM medium/5% FCS per well and insert2.

___________1 PBS+: phosphate buffered saline containing 1.8 mM CaCl2 and 3.98 mM MgSO42 The total medium volume was splitted between insert and well, so that hydro-dynamic equilibrium was achieved between both compartments.


150.000 human foreskin keratinocytes in 100 μl KBM basalmedium/ 5% FCS were subsequently seeded on top of the collagen gel. After 1-2 h incubation at 37°C, a total of 2.5 mlKBM basal medium containing FCS (5%), hEGF (0.1 μg/500 ml) and BPE (15 mg/ 500 ml) was added to each insert andwell. During submersed cultivation medium was exchangedevery day, thereby gradually lowering the FCS concentrationfrom 5% to 0%. After submersed culture the inserts carrying theskin equivalents were transferred into the wells of a conventional 12 well plate or a 12 well ThinCert™Plate. KBM medium with1.88 mM CaCl2 was filled into each well up to the level of the insert membrane (air-lift culture). The air-lift culture was performed for up to 13 days with one medium exchange at day 7.

Determination of the glucose content in cell culture medium

Medium samples from squamous keratinocyte cultures weredrawn after 3, 7, 11, 15 and 19 days in culture (always prior tomedium exchanges). The glucose content of the medium samples was determined using an Accu-Chek® Aviva test fromRoche.

Histological analysis

Reconstructed buccal epithelia were snap frozen over liquid nitrogen embedded in Tissue-Tek® at cultivation day 29. Subsequently cryo-sections of 8 μm thickness were preparedand fixed with acetone (10 min, room temperature). Skin models were subjected to histological analysis after 3, 6, 10 or13 days in air-lift culture. For this purpose, the tissue sampleswere fixed for 1 h with Bouin’s solution, dehydrated and paraffin embedded according to standard protocols. Sections of3 μm thickness were prepared. All tissue samples were stainedwith Hematoxylin and Eosin applying standard protocols.

Figure 1: Steps involved in the reconstruction of a full thickness skin model in ThinCert™ Cell Culture Inserts.A collagen gel containing primary dermal fibroblasts is poured onto the porous membrane of a ThinCert™ Cell Culture Insert (A). Subsequently, this dermis equivalentis cultivated under submersed conditions with cell culture medium reaching above the tissue (B). Primary epidermal keratinocytes are seeded on top of the dermisequivalent. After several days in submersed culture, the medium volume is lowered to the level of the membrane (C). The air-lift culture enables an epidermis withstratum corneum to form (D). For improved nutrient supply the air-lift culture is performed in a deep-well ThinCert™Plate to increase medium volume (C, D).

Multiwell plate

ThinCert™ Cell Culture Insert

ThinCert™Plate

A B C D

Immunohistochemical analysis

Immunhistochemical analysis was performed on 3 μm paraffinesections of skin models cultivated for 10 or 13 days in air-liftculture. Anti Cytokeratin 5/6, Cytokeratin 10 and Filaggrin antibodies were applied at concentrations of 1:1000, 1:2000and 1:400, respectively. Immunohistochemistry was performed according to the instructions of the antibody manufacturers. For the primary antibody detection the HRP detection systemEnVisionTM + was used.

Results

In comparison to monolayer cell culture, tissue reconstructionrequires cell culture in multiple layers at high densities. Hence, specific demands on the applied cultivation systemarise, including an improved nutrient supply and enhanced gas exchange. In ThinCert™ Cell Culture Inserts tissue may be cultivated at the air-liquid interface with direct oxygen supply.With the upper insert compartment unavailable for medium deposition during the air-lift culture, the enlarged well size of theThinCert™Plate provides a large medium and nutrient reservoirsource directly below the insert membrane (Figure 1C and D).

Here, oral squamous keratinocytes were cultivated in air-liftculture using ThinCert™ Cell Culture Inserts in combination withconventional multi-well plates or ThinCert™Plates. Due to thelimited medium reservoir of conventional multi-well plates, it wasnecessary to exchange the cell culture medium every day inthese plates. In contrast, ThinCert™Plates allowed an expanded timeframe for medium exchanges to every 4th day.The improved nutrient supply achieved in ThinCert™Plates waswell reflected in the glucose concentration of the culturemedium, which was higher in samples drawn from ThinCert™Plates than in samples from conventional plates at all examinedstages (3, 7, 11, 15 and 19 days in culture, Figure 2A). As a result the cultivation in the ThinCert™Plate yielded a better tissue quality with thicker tissue and more cell layers (Figure2B).

As mentioned above, air-lift culture not only guarantees properoxygen supply of the cultivated tissue, it also provides anindispensable differentiation stimulus for the formation ofterminal structures of air-exposed tissues such as the stratumcorneum of skin.

Here, the cultivation of a full thickness skin model was used tofurther illustrate the capabilities of the ThinCert™ Cell Culturesystem. The dermis equivalent was first allowed to grow undersubmersed conditions for one week using ThinCert™ Cell Culture Inserts in conventional multiwell plates. The epidermisequivalent was then allowed to differentiate at the air-liquid interface for two weeks using ThinCert™ Cell Culture Inserts inThinCert™Plates.

Figure 2: Improved nutrient supply with the ThinCertTMPlate.A buccal mucosa like epithelium was reconstructed from oral squamous cellsusing ThinCert™ Cell Culture Inserts in combination with either conventional multiwell plates or the ThinCert™Plate over a cultivation period of 29 days. The enlarged medium reservoir of the ThinCert™Plate allowed the reduction of medium changes from every day, as required by the conventional plate, to every 4th day. In addition to reducing the number ofmedium changes, a significantly higher glucose concentration was maintained in the ThinCert™Plate as compared to the standard condition (A, glucose measurements were performed prior to medium changes). Moreover, tissue generated in the ThinCert™Plate was thicker in appearance and contained more cell layers in comparison to tissue generated in the conventional plate (B, Hematoxylin-Eosin staining of thebuccal mucosa epithelium after 29 days in culture).


B

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In contrast, Filaggrin was only detectable in the skin modelscultivated in the ThinCert™Plate (Figure 4C), whereas thosecultivated in the conventional plate were devoid of Filaggrin expression (Figure 4F).

The early onset of Filaggrin expression in the ThinCert™Platesuggests that culture conditions in this plate can promote andaccelerate keratinocyte differentiation during the air-lift culture.At cultivation day 13 Filaggrin became also detectable in the skinmodels cultivated in the conventional plate (data not shown),thus indicating that terminal differentiation was slightly delayed,but not abolished in this control experiment.

Conclusion

The applications presented within this paper illustrate the multiple advantages and possibilities of ThinCert™ Cell Culture products for organotypic tissue culture and tissue reconstruction. The protocols provide detailed instructions thatcan easily be adapted to suit research interests other thanbuccal mucosa and skin reconstruction.

For additional information and continuative literature please referto the website of Greiner Bio-One.

www.gbo.com/bioscience/thincert

Figure 3: Maturation of a skin model cultivated in ThinCert™ Cell CutureInserts and the ThinCert™Plate.The skin model was cultivated at the air-liquid interface and histologicallyanalysed at cultivation days 3 (A), 6 (B), 10 (C) and 13 (D) using Hematoxylin-Eosin staining. First signs of cornification were detectable at cultivation day 6(B). After 10 (C) and 13 (D) days a multi-layered epidermis with well definedstratum corneum had formed.


Histological analysis of the well stratified skin models revealedthe first tendency of cornification at cultivation day 6 (Figure 3B).After 10 and 13 days in air-lift culture a multi-layered epidermiswith defined stratum corneum had developed (Figure 3C andD), thus strongly resembling the stratified structure of nativehuman skin. It is noteworthy that one medium exchange at day7 was sufficient to provide the growing skin model with sufficient nutrients for the entire air-lift cultivation of two weeks.

For characterisation of the differentiation status of the cultivated skin models immunohistochemistry was performedat day 10 of the air-lift culture using antibodies against the earlyterminal differentiation markers Cytokeratin 5/6 and 10 and thelate differentiation marker Filaggrin.

For comparison two sets of skin models were analysed –one using ThinCert™ Cell Culture Inserts in a conventional cellculture plate and one using ThinCert™ Cell Culture Inserts inthe ThinCert™Plate for air-lift culture. Cytokeratin 5/6 and 10were detectable in all skin models independent of the used plate(Figure 4A, B, D, E).

A

C

B

D Stratum corneum

Epidermis

Dermis

50 μm

References

Asselineau D, Bernhard B, Bailly C, Darmon M. (1985) Epidermal morphogenesisand induction of the 67 kD keratin polypeptide by culture of human keratinocytesat the liquid-air interface. Exp Cell Res. Aug;159(2):536-9.

Bell E, Ivarsson B, Merrill C. Production of a tissue-like structure by contraction ofcollagen lattices by human fibroblasts of different proliferative potential in vitro.(1979) Proc Natl Acad Sci U S A. Mar;76(3):1274-8.

Naughton GK, Jacob L, Naughton BA. (1989) A Physiological Skin Model for InVitro Toxicity Studies. Alternative Methods in Toxicology. Vol. 7, ed. A.M. Gold-berg, Mary Ann Liebert, New York: 183-189.

Ponec M, Weerheim A, Kempenaar J, Mommaas AM, Nugteren DH. (1988) Lipidcomposition of cultured human keratinocytes in relation to their differentiation.J Lipid Res. Jul;29(7):949-61.

Sundqvist K, Kulkarni P, Hybbinette SS, Bertolero F, Liu Y, Grafström RC. (1991)Serum-free growth and karyotype analyses of cultured normal and tumorous(SqCC/Y1) human buccal epithelial cells. Cancer Commun. 3(10-11):331-40.


Germany (Main office)Greiner Bio-One GmbHPhone: (+49) 7022 948-0E-Mail: [email protected]

AustriaGreiner Bio-One GmbHPhone: (+43) 7583 6791-0E-Mail: [email protected]

BelgiumGreiner Bio-One N. V.Phone: (+32) 2-4 61 09 10E-Mail: [email protected]

NetherlandsGreiner Bio-One B. V.Phone: (+31) 172-42 09 00E-Mail: [email protected]

FranceGreiner Bio-One SASPhone: (+33) 169-86 25 50E-Mail: [email protected]

UKGreiner Bio-One Ltd.Phone: (+44) 1453-82 52 55E-Mail: [email protected]

JapanGreiner Bio-One Co. Ltd.Phone: (+81) 3-35 05-88 75E-Mail: [email protected]

USAGreiner Bio-One North America Inc.Phone: (+1) 704-261-78 00E-Mail: [email protected]

Revision: March 2008 - 074 062

Patents pending

In part, the skin reconstruction procedure described above is patent-protected(DE 10062623 B4, WO 0192477 A2).

____________________________________________________________________

Acknowledgement

We thank Dr. Dirk Dressler (BioTeSys GmbH/Esslingen/Germany) for providing ex-perimental data from squamous cell cultures. In addition, we thank Dr. Michaela Weimer and Mrs. Sybille Thude (Fraunhofer Institute for InterfacialEngineering and Biotechnology/Stuttgart/Germany) for their help with the cultivation of skin models.

Figure 4: Immunohistochemical characterisation of skin models.Skin models were cultivated in ThinCert™ Cell Culture Inserts and a conventional cell culture plate (A–C) or a ThinCert™Plate (D–F) and immunohistochemically analysedat cultivation day 10 using antibodies against Cytokeratin 5/6 (A, D), Cytokeratin 10 (B, E) and Filaggrin (C, F). The positive staining for the early differentiation markers Cytokeratin 5/6 (entire epidermis in A, D) and Cytokeratin 10 (suprabasel epidermal layers in B, E) indicates the onset of keratinocyte differentiation in both cell culture plates prior to cultivation day 10. Only the skin model that has been cultivated in the ThinCertTMPlate showed immunoreactivity againstthe late differentiation marker Filaggrin (Stratum granulosum in F), thus indicating that in the ThinCertTMPlate keratinocyte differentiation was more advanced than in thecontrol plate (no immunoreactivity in C).

BA C

D E F

50 μm


Application NoteThe reconstruction and immunocytochemical characterisationof polarised epithelia in ThinCert™ cell culture inserts


Alexa Fluor 488 goat antirabbit IgG antibody


Alexa Fluor 546 goat antimouse IgG antibody


Rabbit Anti Claudin-1 antibody Zytomed Systems GmbH RP153

Mouse Anti E-Cadherin antibody Becton Dickinson GmbH 610181

Rabbit Anti ZO1 antibody Invitrogen GmbH 40-2300, PAD:ZMD.437

CELLSTAR® 24 wellcell culture plate


DakoCytomation FluorescentMounting Medium

Dako Deutschland GmbH S3023

DAPI, dilactate Sigma-Aldrich Chemie GmbH D9564

DMEM medium Biochrom AG F0435

Fetal calf serum Invitrogen Life Technologies 10270-106

Fibronectin TeBu-Bio GmbH 2004

Formalin Sigma-Aldrich Chemie GmbH HT5014

L-alanyl-L-glutamine Biochrom AG K0302

MEM-amino acids, 50x Biochrom AG K0363

PBS Biochrom AG L1825

RPMI medium Biochrom AG F1295

ThinCert™ 24 well cell cul-ture insert with 0.4 µm poresand transparent membrane


ThinCert™ 24 well cell cul-ture insert with 0.4 µm poresand translucent membrane


Triton® X100 Sigma-Aldrich Chemie GmbH T8787

Water (tissue culture grade) Sigma-Aldrich Chemie GmbH W3500

Introduction

The establishment of biologically relevant in vitro approachesis becoming increasingly important as objections to animalexperimentation continue to rise. One of the challenges facingthe composition of such models is to reconstruct epithelia ortissues in such a way that would preserve their native biolo-gical features and functions; to include the occurrence ofpolarisation, multi-layered growth, barrier function andvectorial transport.

Cell culture inserts with porous membrane supports are widelyaccepted tools for the reconstruction of functional epitheliaand tissues in vitro. In cell culture inserts, high density cellpopulations may be achieved with nourishment from twodifferent areas: the surface that faces the porous membranesupport as well as the one that faces away. This dual nutrientaccess, along with the presence of a solid substrate that maycarry components of the ECM1, has proven to be an indis-pensable stimulus to establish polarity in cultivated epithelia(Chambard et al., 1983; Guguen-Guillouzo and Guillouzo,1986; Saunders et al., 1993).

In the experiments presented, epithelia were cultivated onThinCert™ cell culture inserts using Madin-Darby caninekidney cells (MDCKII) and colorectal adenocarcinoma cells(CACO-2). Applying fluorescence immunocytochemistry thecell adhesion protein E-Cadherin and the tight junctionproteins Claudin-1 and ZO-1 were localised to the basolateralcompartment and the apical rim of the cell membrane,respectively. The localisation of these proteins to sides wherethey would also occur under in vivo conditions (see Fig. 1)clearly demonstrates the suitability of ThinCert™ cell cultureinserts for the cultivation of epithelia and the achievement ofcellular polarity in vitro. Within this application note, detailedprotocols for the cultivation of epithelial cells and their mole-cular characterisation by fluorescence immunocytochemistryare provided using ThinCert™ cell culture inserts.

Material and Methods

Coating of cell culture inserts

For all the experiments described here, 24 well ThinCert™ cellculture inserts with 0.4 µm pores and transparent or trans-lucent PET membranes were used. The inserts were placed inthe wells of a CELLSTAR® 24 well cell culture plate. 60 µl of anaqueous fibronectin solution (50 µg/ml) was applied to eachcell culture insert. After 2 h incubation at room temperature,the fibronectin solution was removed and the cell cultureinserts were rinsed three times with PBS.

___________1 ECM: extracellular matrix

Figure 1When cultivated on porous membrane supports (ThinCert™) epithelial cellsform a dense cell layer. The formation of tight junctions at the apical rim ofthe lateral cell membrane and the establishment of basolateral and apicalmembrane compartments prove this cell layer to be an epithelium with itscharacteristic features.

Material


1. Remove cell culture medium from insert

2. Fix cells for 5 min on ice with 4% formalin, use 500 µl per insert

3. Wash insert twice with PBS, use 500 µl per insert

4. Permeabilise cells for 25 min with 0.5% Triton in PBS, use 500 µl perinsert

5. Wash insert twice with PBS, use 500 µl per insert

6. Block unspecific binding sites for 1.5 h with 10% FCS, use 500 µlper insert

7. Wash inserts three times for 5 min with PBS, use 500 µl per insert

8. Incubate cells for 1 h with primary antibody in 1% FCS, use 100 µlper insert

9. Wash inserts three times for 5 min with PBS, use 500 µl per insert

10. Incubate cells for 1 h with secondary antibody in 1% FCS, use100 µl per insert

11. Wash inserts two times for 5 min with PBS, use 500 µl per insert

12. Incubate cells 5 min with DAPI in PBS (10 µg/ml), use 100 µl perinsert

13. Wash inserts two times for 5 min with PBS, use 500 µl per insert

14. Detach membrane from the insert housing using a scalpel

15. Mount membrane onto a microscopy slide, use fluorescenceembedding medium

Cell culture

Cell cultures were prepared and maintained according tostandard cell culture procedures. For the propagation ofMDCKII cells DMEM medium with 10% fetal calf serum (FCS)and 4 mM L-alanyl-L-glutamine was used. CACO-2 cells werecultivated in DMEM medium supplemented with 20% FCS,4 mM L-alanyl-L-glutamine and 1x MEM-amino acids.

For the establishment of an epithelial cell layer 5x103, 25x103,5x104 or 4x105 cells (MDCKII or CACO-2) were sown intoeach cell culture insert and cultivated for 24, 48 and 72 h ina cell culture incubator (37°C, 5% CO2).

Immunocytochemistry2

Unless otherwise specified, all steps were carried out at roomtemperature. Prior to fixation, the cell culture medium wasremoved from both cell culture inserts and cell culture platewells. 500 µl 4% formalin solution was added to each insert3.The fixation was carried out for 5 min on ice, followed by twowashes with 500 µl PBS per insert. Cells were permeabilisedfor 25 min using 500 µl 0.5% Triton/PBS per insert, followedby two washes with 500 µl PBS per insert. For the blocking ofnon-specific protein binding sites, 500 µl 10% FCS/PBS wasapplied to each insert and incubated for 1.5 h. Thereafter, theinserts were washed three times 5 min with 500 µl PBS. Afterremoving the washing solution, 100 µl primary antibodysolution in 1% FCS/PBS was applied to each insert, andan incubation of 1 h was performed4.

For a detailed description of the applied antibodies (origin,specificity and working concentrations), see Tab. 1. Afterincubation with the primary antibodies, the inserts werewashed three times for 5 min with 500 µl PBS. 100 µl of thesecondary antibody solution in 1% FCS/PBS was applied toeach insert and incubated for 1 h at room temperature. Fordetails on the secondary antibodies used in this protocol seeTab. 1. Alexa 488 coupled secondary antibodies were used todetect the ZO1 and Claudin-1 specific primary antibodies.Alexa 546 was used to detect the anti E-Cadherin primaryantibody. After incubation with the secondary antibodies, twoadditional washing steps with 500 µl PBS per insert, 5 minincubations, were performed. For the subsequent nuclearstaining, each insert was incubated for 5 min with 100 µl DAPIsolution (10 µg/ml). Thereafter, the inserts were washed twotimes for 5 min with 500 µl PBS. The insert membranes weredetached from the insert housings using a scalpel (see Fig. 3)and mounted onto microscopy slides using fluorescencemounting medium.

Table 1: Key features of the applied antibodies

___________2 For a flow chart of the immunocytochemistry protocol see Fig. 23 At this stage, solution was not added into the lower compartment (well of the plate). For fixative to remain in the upper compartment (insert), it is necessary to keep the

lower compartment dry to ensure a liquid bridge does not form between the membrane underside and the plate well bottom. This also applies to all subsequent stepswhere solution is transferred solely into the upper compartment.

4 Alternatively, some primary antibody incubations were performed overnight at 4°C.

Figure 2Flow chart. This guide is for quick reference only. Familiarise yourself withdetails before performing the assay. All volumes are adjusted to fit 24 wellinserts and have to be modified if other inserts are used.

Designation Origin Coupledfluoro-phore

Detectedprotein

Workingconcentration[dilution]

Pri

mar

yan

tibo

die

s

anti E-Cadhe-rin antibody

mouse - humanE-Cadherin

2.5 µg/ml[1:100]

anti ZO1antibody

rabbit - human Zonaoccludens 1protein

2.5 µg/ml[1:100]

anti Claudin-1antibody

rabbit - humanClaudin-1protein

~10 µg/ml[1:100]

Sec

ond

ary

antib

od

ies

Alexa 546anti mouseIgG antibody

goat Alexa 546 mouse IgG 8 µg/ml[1:250]

Alexa 488anti rabbitIgG antibody

goat Alexa 488 rabbit IgG 8 µg/ml[1:250]


Microscopy

Images were captured with a Zeiss Axioplan 2 wide fieldfluorescence microscope. Z-scans were acquired with a ZeissCell Observer with Apotome applying two times averaging.Pictures were processed with the AxoVision 4.5 software(Zeiss) and mounted using Photoshop 6.0 (Adobe). Details ofthe applied filter sets are given in Tab. 22.

Results

Optimum cell culture conditions for the formation of tight junctions

In order to determine the optimum cell culture conditions forthe formation of epithelium-like cell layers, MDCKII cells werecultivated on ThinCert™ cell culture inserts at various seedingdensities for different time periods. Cell cultures were fixedand stained for E-Cadherin and the tight junction proteinClaudin-1 according to the above-mentioned protocol.

Seeding 5x103 cells per insert and keeping them 24 h in culture did not allow the cells to reach confluence andClaudin-1 expression above background levels (Fig. 4, left column, lower panel). Although confluence was reached after48 h Claudin-1 expression was still low at this time (Fig. 4, leftcolumn, center panel). After 72 h the cells grew to a maximumdensity and began to express high levels of Claudin-1 (Fig. 4,left column, upper panel).

With higher initial seeding densities (25x103 and 5x104 cellsper insert) confluence and expression of Claudin-1 wereachieved after only 24 h in cell culture (Fig. 4, center and rightcolumn). Under all conditions, the localisation of E-Cadherin to the cell membrane correlated with the appearance of Claudin-1 (Fig. 4, all columns). It is noteworthy, that with an increasing cell density also the ratio between the nuclear andcytoplasmic volumes increased (Fig. 4, all columns). This phenomenon correlates with cells and nuclei forming a smallbase area and achieving a tall appearance (not shown), thusindicating the cell layer obtained a high prismatic morphology.

In summary it may be recommended to seed cells at a highdensity (5x104 cells per insert and above) to achieve a rapidformation of tight junctions in vitro.

Figure 3For the detachment of the capillary pore membrane from the insert housing a scalpel is plunged into the membrane adjacent to the insert housing (A).The membrane is cut out moving the scalpel alongside the inner edge of the housing (B), thereby leaving a small segment attached to the membrane (arrowhead in C). Finally, using tweezers, the membrane is torn off the housing (C-E). The membrane is now ready for further processing such asmounting onto a microscopy slide, sectioning or other applications.

Fluorophores Excitationwavelength

Emissionwavelength

Wavelengthtransmittedthroughbeamsplitter

DAPI (4',6-diami-dino-2-phenyl-indole)

335-383 nm 420-470 nm > 395 nm

Alexa Fluor 488 455-495 nm 505-555 nm > 500 nm

Alexa Fluor 546 533-558 nm 570-640 nm > 570 nm

Table 2: Features of the used fluorescence filters sets

Establishment of polarity in cultivated epithelial cells

In another series of experiments MDCKII or Caco-2 cells were seeded at a high initial density (4x105 cells per insert),cultivated for 48 h and stained simultaneously for the tightjunction proteins Claudin-1 or ZO1 and the basolateral cell adhesion protein E-Cadherin. Nuclei were counterstained withDAPI. Image series along the z-axis5 of the stained cell layerswere acquired using a Zeiss Cell Observer with Apotome. Individual images were aligned to a z-stack, and virtual x-z-and y-z-cross sections along lines of interest (see Figs. 5 and6) were generated using the AxoVision 4.5 software (Zeiss).

___________5 Here, the z-axis corresponds to the apical-basal-axis of the cultivated epithelium.


The cultivation and analysis of MDCKII and CACO-2 cellsaccording to this protocol revealed the formation of tightjunctional complexes in the upper (apical) part of the lateralcell membrane (Figs. 5 and 6). These tight junctions seal thecell membrane, thus generating apical and basolateral mem-brane compartments. Whereas E-Cadherin was detectablein the entire basolateral membrane compartment, the apicalmembrane compartment was devoid of E-Cadherin in all cells(Figs. 5 and 6). In contrast to MDCKII cells CACO-2 cellsformed a less regular network. Projections of the stainingpatterns of Claudin-1, E-Cadherin and DAPI along the z-axisdid not align as well as those observed with MDCKII cells.This phenomenon is mainly due to the rather irregular thanprismatic pattern of the CACO-2 cell layer. However, theanalysis of the Claudin-1 and E-Cadherin distribution alongthe z-axis clearly indicated the formation of tight junctions inthe apical part of the lateral cell membrane and the separationof the membrane into apical and basolateral compartmentswithin these cells (Fig. 6B).

Figure 4Influence of the initial seeding density and the cultivation time on theexpression of Claudin-1 and E-Cadherin. With a low initial seeding density(5x103 cells per insert) up to 72 h of cell culture are required before Claudin-1expression and the localisation of E-Cadherin to the cell membrane becomedetectable (left column). With higher seeding densities (25x103 and 5x104

cells per insert) Claudin-1 expression and the localisation of E-Cadherin tothe cell membrane become detectable after only 24 h in cell culture (centerand right column).

Figure 5Microphotographs of MDCKII cells that were processed for fluorescenceimmunocytochemistry against E-Cadherin (red channel) and ZO1 (greenchannel). Nuclei were stained with DAPI (blue channel). Serial photographswere acquired along the z-axis of the cell layer (from apical to basal) and alignedto form a stack of pictures (z-stack). The large quadratic images in A-Crepresent one individual photograph that has been chosen from an apical levelof the z-stack. Using the Axio Vision 4.5 software, the z-stack was sectionedalong two planes, each of them being indicated by a gray line. Sectioning alongthe horizontal line produced the x-z-cross section depicted on top of eachpanel, sectioning along the vertical line produced the y-z-cross section shownat the right margin of each panel.

In the x-z-cross section as well as in the y-z-cross section the ZO1 positive beltof tight junctions appears in an apical domain of the lateral cell membrane (B,C). This domain clearly marks the site at which a basolateral compartment withE-Cadherin expression is separated from an apical compartment withoutE-Cadherin expression (A, C).


Conclusion and Discussion

In these experiments, evidence is provided that MDCKII andCACO-2 cells establish a polarised epithelium when cultivatedon ThinCert™ cell culture inserts. Thus, the formation of ZO1and Claudin-1 positive tight junctions was observed in anapical domain of the lateral cell membrane, whereas E-Cad-herin expression was detected in the basolateral membranecompartment below.

In general ThinCert™ cell culture inserts were found to beexcellent tools for fluorescence immunocytochemical stain-ings. The overall low autofluorescence of the ThinCert™membrane assures low background signals6. In some casesenhanced light scattering may be observed with translucentmembranes. If so, special care should be taken to work withoptimised immunocytochemistry protocols, to yield highlyspecific fluorescence signals. No differences in the perform-ance of translucent and transparent membranes were ob-served during the course of these experiments.

In this study, specific protein localisation was exploited todemonstrate epithelial polarisation on ThinCert™ cell cultureinserts. Similarly, epithelial polarisation was depicted inKopplow et al., 2005 by showing the specific localisation oftransporter proteins to basolateral or apical membranecompartments. In addition, functional aspects of epithelialpolarisation on ThinCert™ cell culture inserts have beendescribed, such as the vectorial transport (Kopplow et al.,2005; Letschert et al., 2005) and the polarised endocytosis(Mettlen et al., 2006). In summary, ThinCert™ cell cultureinserts prove to be excellent tools for the reconstruction ofepithelia and the restoration of their biological functions invitro.


Figure 6MDCKII (A) and CACO-2 cells (B) were processed for fluorescence immuno-cytochemistry to detect E-Cadherin (red channel) and Claudin-1 (greenchannel). Nuclei were stained with DAPI (blue channel). For a detailedexplanation of the image acquisition and processing procedures see legendto Fig. 5.In both, MDCKII (A) and CACO-2 cells (B) Claudin-1 immunoreactivity islocalised to an apical domain of the lateral cell membrane. E-Cadherinimmunoreactivity is only found in the basolateral membrane compartmentbelow the Claudin-1 positive belt of tight junctions.

References

Chambard M, Verrier B, Gabrion J, Mauchamp J. (1983)Polarization of thyroid cells in culture: evidence for the baso-lateral localizationof the iodide “pump” and of the thyroid-stimulating hormone receptor-adenylcyclase complex. J Cell Biol. Apr;96(4):1172-7.

Guguen-Guillouzo C, Guillouzo A. (1986) Isolated and cultured hepatocytes.Paris, Les Éditions INSERM, John Libbey Eurotext:1-12

Kopplow K, Letschert K, Konig J, Walter B, Keppler D. (2005) Human hepato-biliary transport of organic anions analyzed by quadruple-transfected cells.Mol Pharmacol. Oct;68(4): 1031-8.

Letschert K, Komatsu M, Hummel-Eisenbeiss J, Keppler D. (2005) Vectorialtransport of the peptide CCK-8 by double-transfected MDCKII cells stablyexpressing the organic anion transporter OATP1B3 (OATP8) and the exportpump ABCC2. J Pharmacol Exp Ther. May;313(2):549-56.

Mettlen M, Platek A, Van Der Smissen P, Carpentier S, Amyere M, Lanzetti L,de Diesbach P, Tyteca D, Courtoy PJ. (2006) Src triggers circular ruffling andmacropinocytosis at the apical surface of polarized MDCK cells. Traffic.May;7(5):589-603.

Saunders NA, Bernacki SH, Vollberg TM, Jetten AM. (1993) Regulation of trans-glutaminase type I expression in squamous differentiating rabbit tracheal epithe-lial cells and human epidermal keratinocytes: effects of retinoic acid and phorbolesters. Mol Endocrinol. Mar;7(3):387-98.

____________________________________________________________________

Acknowledgement

We thank Dr. Katalin Schlett (Eotvos Lorand University/Budapest/Hungary)and Dr. Gertrude Bunt (University Stuttgart/Germany) for their help with theacquisition and processing of the presented images.

___________6 For this also compare Fig. 5 where x-z- and y-z-cross sections include parts of the ThinCert™ membrane.

Germany (Main office)Greiner Bio-One GmbH

Phone: (+49) 7022 948-0

E-Mail: [email protected]

AustriaGreiner Bio-One GmbH

Phone: (+43) 7583 6791-0


BelgiumGreiner Bio-One N. V.

Phone: (+32) 2-4 61 09 10


NetherlandsGreiner Bio-One B. V.

Phone: (+31) 172-42 09 00


FranceGreiner Bio-One SAS

Phone: (+33) 169-86 25 50


UKGreiner Bio-One Ltd.

Phone: (+44) 1453-82 52 55


JapanGreiner Bio-One Co. Ltd.

Phone: (+81) 3-35 05-88 75


USAGreiner Bio-One North America Inc.

Phone: (+1) 704-261-78 00


Revision: March 2007 - 073 100

forumT e c h n i c a l N o t e s a n d A p p l i c a t i o n s f o r L a b o r a t o r y W o r k

No. 8, 2007

Content

1. Characteristics of ThinCertTM cell culture inserts

2. Cell-based assays in ThinCertTM cell culture inserts

2.1 Migration and invasion

2.2 Co-culture

2.3 Ephitelia, immunocyto-

chemistry and transport

studies

2.4 Organotypic culture and

air-lift-culture

3. Continuative literature


ThinCert™ cell culture products –innovative solutions for cell-based assays and tissue culture

Ethical, scientific and financial concerns lead to an

increasing importance of cell-based assays and three-

dimensional cell culture in lieu of live-animal experimentation.

Therefore, new cell culture devices have been developed

that allow the establishment of an in vitro environment

with maximum retention of native functionality. With

respect to these developments, ThinCert™ cell culture

inserts with porous PET membranes are significant

because they form a two-compartment system to readily

mimic a variety of in vivo situations, such as:

� migration and relocation of cells (Fig. 1A),

� interaction and communication of physically separated

cell populations (Fig. 1B),

� formation of tight cell-cell junctions,

� vectorial transport between two lumens (Fig. 1C),

� tissue growth and differentiation at the

air-liquid-interface (Fig. 1D).

forum No. 8, 2007

1. Characteristics of ThinCert™ cell culture inserts

ThinCert™ cell culture inserts combine consistent high

quality with a user-friendly format. For example, the

inserts are specifically designed to entail an eccentric

position in the wells of a multiwell plate thereby

facilitating pipette access to the lower compartment

(Fig. 2A). The insert housing and PET membrane are

combined by a thermobonding process which creates

a very robust seal that prevents any membrane leakage.

Nevertheless, the insert membrane can be cut out and

easily subjected to downstream processing, such as

sectioning and mounting on microscopy slides (Fig. 2B).

The capillary pores of the ThinCert™ membrane are

produced by a precise track-etching process which re-

sults in an even pore distribution and in highly uni-

form pore diameters (Fig. 2C and D).

2. Cell-based assays in ThinCert™ cell culture inserts

The following application examples illustrate the usage

of ThinCert™ cell culture inserts in diverse cell-based

assays. A selection guide for the appropriate insert

type for your application of choice is provided in Table 1.

2.1 Migration and invasion

Cell migration plays a significant role in physiological

and pathological processes during embryonic

development, wound healing, immune response,

inflammation, and tumorigenesis. The filter assay or

‘Boyden chamber assay’ (Boyden, 1962) is a classical

in vitro model used to study cell migration. This assay

is performed in cell culture inserts with porous

membrane supports (e.g. ThinCert™) and involves cell


Figure 1: Overview on applications of ThinCert™ cell culture insertsA, B, C: Schematic drawings illustrating experimental setups formigration and invasion assays (A), co-cultivation (B) and transportstudies (C). D: Tissue growth at the air-liquid-interphase.

Figure 2: Characteristics of ThinCert™ cell culture insertsA: Specific design of ThinCert™ cell culture inserts with easy pipette accessto the lower compartment. B: Detachment of a ThinCert™ membranewith a scalpel. C: Microscopic view of a porous ThinCert™ membranewith even pore distribution. D: Pore size distribution of different porousmembranes with nominal pore sizes of 0.4 μm.

A B C

D

Upper compartment

Lower compartment

Cells

Epithelium Tissue

Porous membrane

Plate

ThinCertTM cell culture insertA B

C D

forum No. 8, 2007

migration from the upper compartment through the

pores of the membrane towards a chemo-attractant

source in the lower compartment.

To determine the invasive potential of cells, the

membrane may also be coated with an extracellular

matrix (ECM), which mimics the basal lamina (Albini et

al., 1987). Usually ThinCert™ cell culture inserts with

8.0 μm pores are used in such assays.

Invasive cells (e.g. HT1080) and non-invasive cells

(e.g. MCF7, NIH3T3) may be distinguished based on

their invasion index, which is defined as the ratio of the

number of cells migrating in the presence and absence

of an ECM coating (Fig. 3A).

Several procedures may be conducted in order to

quantify migratory cells in the filter assay. For example,

cells may be stained with crystal violet, non-migratory

cells may be removed from the upper compartment

with a cotton swab and migratory cells may be counted

with a microscope (Hiscox et al., 2006). Other procedures

involve cell staining with fluorescent dyes such as

DAPI or Calcein-AM , the detachment of migratory

cells from the underside of the membrane using

Trypsin-EDTA and their subsequent quantification

using a fluorescence plate reader.

2.2 Co-Culture

Co-culture describes a rather heterogeneous field

including applications as diverse as the investigation

of immune cell interactions, the stimulation of cell

proliferation, the maintenance of cell differentiation and

the restoration of heterocellular functions in vitro (e.g.

blood-brain-barrier). With ThinCert™ cell cultures inserts,

cells may be seeded in the upper and lower

compartment, which keeps them physically separated

at all stages of the co-culture. The pores of the ThinCert™

membrane, however, allow the exchange of molecules

between the two compartments and hence between

the two cell populations.

MCF7 cells cultivated on ThinCert™ cell culture inserts

with translucent membranes and 0.4 μm pores may

receive a growth promoting stimulus from primary

human fibroblasts cultivated on the underside of the

membrane. This enhanced proliferation is indicative of

an increased fraction of proliferative, Ki67 expressing

cells in proportion to the total cell number (Fig. 3B).

As mentioned above, it is important to note that cells

co-cultivated in the lower compartment may be grown

not only on the surface of the multiwell plate, but also

on the underside of the membrane. Therefore, the

distance between the two cell populations can be

quite close (in the range of the diameter of a single

cell). Both sides of the membrane are tissue culture

treated to encourage this possibility.


Figure 3: Overview on applications ofThinCert™ cell culture insertsA: In ECM coated inserts invasiveHT1080 cells reveal a higher invasionindex towards an FCS source in thelower compartment than non-invasiveMCF7 and NIH3T3 cells. B: MCF7 cellswere grown on a 0.4 μm pore membraneand obtained a proliferative stimulus fromhuman fibroblasts co-cultivated in thelower compartment. Non-proliferativecells were identified by lack of Ki67immunofluorescence (arrows). C: Fortransport studies epithelia may begenerated from MDCK II cells on 0.4 μmThinCert™ cell culture inserts. Immuno-fluorescence indicates the polarisedlocalisation of Claudin-1 in the tightjunctions and E-Cadherin basolaterally.

A B

DAPIKi67

C

Claudin-1 E-Cadherin DAPI

forum No. 8, 2007

2.3 Epithelia, immunocytochemistry and transport

studies

Transport studies are among the most frequent

applications of ThinCert™ cell culture inserts. Here,

the goal is to reconstruct a functional epithelium from

individual cells and to study the active transport of

substances from one compartment through the

epithelium to the other compartment. It has been

shown that polarised epithelia with tight junctions and

basolateral and apical membrane compartments can

be generated in cell culture inserts with porous

membrane supports. The interaction of two

phenomena seems to account for this effect: (1) the

insert membrane, which usually carries an ECM

treatment, can mimic the basement membrane; and

(2) the pores of the membrane allow the cells to take

up nutrients from the basolateral side (Chambard et al.,

1983; Guguen-Guillouzo and Guillouzo, 1986; Saunders

et al., 1993). Again, it is the similarity between the insert

system and the in vivo situation that maintains the native

cell function in vitro.

One prerequisite for a successful transport study is the

establishment of a tight epithelium without trans-cellular

leakage. Several techniques may be used to verify how

tight the cultivated epithelium is, such as the measu-

rement of transepithelial electric resistance (TEER) and

the determination of transcellular leakage with non-

transportable markers (³[H]inulin; Kopplow et al., 2005).

Additionally, some of the prepared cultures may be sa-

crificed and analysed for tight junction formation

using immunofluorescence (Mettlen et al., 2006).

Fig. 3C represents a microphotograph of a MDCK II

cell layer cultivated on translucent ThinCert™ cell culture

inserts with 0.4 μm pores. The insert membrane has

been cut out and subjected to immunocytochemistry.

Confocal fluorescence images have been acquired

and combined to a three dimensional image with red

fluorescence indicating immunoreactivity against the

basolateral marker E-Cadherin and green fluorescence

showing the localisation of the tight junction protein

Claudin-1. Nuclei were counterstained with DAPI.

Polarisation is evident from the localised expression of

E-Cadherin and Caudin-1.

In general, ThinCert™ cell culture inserts with translucent

membranes are recommended for transport assays.

Additional hints on the appropriate membrane quality

and the experimental design are given elsewhere

(Letschert et al., 2005 and continuative literature

resources below).


Figure 4: Tissue cultivation in the ThinCert™PlateA, B: The ThinCertTMPlate comprises extra deep wells and allows largermedium volumes to be applied during tissue culture at the air-liquid-interface. B: Cultivation of a gingival epithelium in ThinCert™ cellculture inserts in the ThinCert™Plate and a conventional multiwell plate.With the ThinCertTMPlate a higher glucose content could be maintainedin the medium.

ThinCertTMPlate Conventional plate

A

B

forum No. 8, 2007

2.4 Organotypic culture and air-lift-culture

Besides the above mentioned applications, ThinCert™

cell culture inserts are widely used to reconstruct

and/or maintain tissue in vitro. In organotypic culture a

tissue, previously isolated from an anesthetised animal,

can be kept alive for prolonged periods (up to several

months). In contrast the term ‘tissue reconstruction’

refers to the de novo generation of a tissue from single

cells utilising cell culture techniques. Both procedures

use cell culture inserts and entail tissue growth at the

air-liquid-interface. The latter allows the cultivated tissue

to reach or maintain the required high cell density without

limitations from gas exchange. Furthermore, for some

tissue types, the direct exposure of the cultivated cells

to the surrounding atmosphere serves as an indispensable

differentiation stimulus. For example, keratinocytes

only form a horny layer (stratum corneum) when exposed

to the surrounding air.

Due to the hanging geometry of most cell culture inserts,

only a little space remains between the insert mem-

brane and the bottom of the multiwell plate. Therefore,

only a small amount of medium is available to the

cultures at the air-liquid-interface. This limited medium

reservoir conflicts strongly with the increased nutrient

consumption of three-dimensional tissues, which

reach cell densities far greater than those of two-

dimensional cell layers. In order to solve this disparity,

Greiner Bio-One developed the ThinCert™Plate – a

novel cell culture plate with extra deep wells (Fig. 4A).

The ThinCert™Plate permits the application of large

medium volumes to cultures at the air-liquid-

interface. Therefore, the frequency of medium changes

is severely reduced.

In the provided application example (Fig. 4 und 5),

gingival epithelia were generated using 12 well ThinCert™

cell culture inserts with 0.4 μm pores in combination

with either conventional 12 well plates or the novel

ThinCert™Plate. The enlarged medium reservoir of the

ThinCert™Plate reduced the medium changes from

every day, as required by the conventional plate, to


every 4th day. In addition to reducing the number of

medium changes, a significantly higher glucose

concentration was maintained in the ThinCert™Plate

compared to the standard conditions. (Fig. 4B).

Moreover, tissue generated in the ThinCert™Plate

appeared thicker and had more cell layers (Fig. 5).

3. Continuative literature

Greiner Bio-One offers several application protocols

for the practical integration of ThinCert™ cell culture

inserts. Each application protocol addresses a specific

question and provides detailed laboratory instructions.

Application protocols and a list of primary literature

implicating ThinCert™ cell culture inserts may be

found on the homepage of Greiner Bio-One under

www.gbo.com/bioscience/thincert.

Figure 5: Tissue cultivation in the ThinCert™Plate Cultivation in the ThinCert™Plate yields an improved tissue quality withmore cell layers (15d/29d: 15/29 days in culture).

A B

C D

20 μm

29d15d

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forum No. 8, 2007


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Germany (Main office): Greiner Bio-One GmbH · (+49) 7022 948-0 · [email protected], Belgium: Greiner Bio-One N. V. · (+32) 24610910 · [email protected], Brazil: Greiner Bio-One Brazil · (+55) 1934 68 9600 · [email protected], China: Greiner Bio-One GmbH · (+86) 21 6272 7058 2051 · [email protected],France: Greiner Bio-One SAS · (+33) 169862550 · [email protected], Japan: Greiner Bio-One Co. Ltd. · (+81) 335058875 · [email protected], Netherlands: Greiner Bio-One B.V. · (+31) 172420900 · [email protected], UK: Greiner Bio-One Ltd. · (+44) 1453825255 · [email protected], USA: Greiner Bio-One North America Inc. · (+1) 7042617800 · [email protected]

References

Albini A., Iwamoto Y., Kleinman H.K., Martin G.R., Aaronson S.A.,Kozlowski J.M., McEwan R.N. (1987) A rapid in vitro assay forquantitating the invasive potential of tumor cells. Cancer Res.Jun 15;47(12):3239-45.

Boyden S. (1962) The chemotactic effect of mixtures of antibodyand antigen on polymorphonuclear leucocytes. J Exp Med. Mar1;115:453-66.

Chambard M, Verrier B, Gabrion J, Mauchamp J. (1983) Polarisation ofthyroid cells in culture: evidence for the baso-lateral localisation of theiodide “pump” and of the thyroid-stimulating hormone receptor-adenylcyclase complex. J Cell Biol. Apr;96(4):1172-7.

Guguen-Guillouzo C, Guillouzo A. (1986) Isolated and culturedhepatocytes. Paris, Les Éditions INSERM, John Libbey Eurotext:1-12

Hiscox S, Morgan L, Green TP, Barrow D, Gee J, Nicholson RI.(2006) Elevated Src activity promotes cellular invasion and motility intamoxifen resistant breast cancer cells. Breast Cancer Res Treat.2006 Jun;97(3):263-74.

Kopplow K, Letschert K, Konig J, Walter B, Keppler D. (2005)Human hepatobiliary transport of organic anions analyzed byquadruple-transfected cells. Mol Pharmacol. Oct;68(4): 1031-8.

Letschert K, Komatsu M, Hummel-Eisenbeiss J, Keppler D. (2005)Vectorial transport of the peptide CCK-8 by double-transfectedMDCK II cells stably expressing the organic anion transporterOATP1B3 (OATP8) and the export pump ABCC2. J Pharmacol ExpTher. May;313(2):549-56.

Mettlen M, Platek A, Van Der Smissen P, Carpentier S, Amyere M,Lanzetti L, de Diesbach P, Tyteca D, Courtoy PJ. (2006) Src triggerscircular ruffling and macropinocytosis at the apical surface ofpolarized MDCK cells. Traffic. May;7(5):589-603.

Saunders NA, Bernacki SH, Vollberg TM, Jetten AM. (1993)Regulation of transglutaminase type I expression in squamousdifferentiating rabbit tracheal epithelial cells and human epidermalkeratinocytes: effects of retinoic acid and phorbolesters. MolEndocrinol. Mar;7(3):387-98.

Table 1: Suitabilityof ThinCert™ mem-branes for differentapplications.+ well suited, - not suited

Catalogue number

Pore size [μm]

Pore density [cm-2]

Membrane opacity

Bright field microscopy

Co-Culture

Culture at the air-liquid-interface

Electron microscopy

Fluorescence microscopy

Immunocytochemistry

Migration/Invasion

Organotypic culture

TEER

Transport studies

657610665610662610

657631665631662631

657640665640662640

657641665641662641

657630665630662630

657638665638662638

0.4

1 x 108

translucent

-+++++-+++

0.4

2 x 106

transparent

++++++-++-

1.0

2 x 106

transparent

++++++-++-

3.0

0.6 x 106

transparent

+-

(+)++++(+)+-

3.0

2 x 106

translucent

--

(+)++++(+)+-

8.0

0.15 x 106

translucent

---++++-+-

Application NoteTransepithelial Electrical Resistance and Impedance Measurements with ThinCert™ Cell Culture Inserts and the cell Zscope® System


Introduction

Barrier-forming cells have become a popular in vitro model to study the paracellular transport of substances. Key to such experiments is the cultivation of endothelial or epithelial cells on permeable membrane supports (e.g. ThinCert™ cell culture inserts). By culturing on porous membranes, these cells develop their specific features that are also found in intact tissues, such as the formation of dense layers with tight cell-cell junctions and established cellular polarity. An excellent tool to assess the barrier function of reconstructed epithelia and endothelia is the transepithelial or -endothelial resistance measurement (TER). TER measurements may be performed on vital cell cultures in a label-free manner. By exceeding certain TER threshold, researchers can conclude that the cell layer is confluent and the formation of tight cell-cell junctions has occurred (Fig. 1).

The classical way of TER measurement is the use of simple handheld devices with chopstick-type electrodes. This setup allows the approximate determination of the ohmic resistance of the barrier-forming cell layer (Fig. 2A). Recently, impedance measurements have gained popularity for the characterisation of epithelial or endothelial cell layers in vitro. The electric impedance of a cell layer can be accurately measured by placing one electrode on each side of the membrane and applying a small AC voltage with varying frequency. The cell related parameters, resistance TER and capacitance CCL, can be deduced from the measured impedance spectra by applying a parametric function and a best fit algorithm [1]. The capacitance CCL may provide additional information about the cell layer’s properties – in particular it is indicative of the formation of membrane protrusions, such as microvilli [2].

This technical note illustrates the preeminent suitability of ThinCert™ cell culture inserts for the reconstruction of barrier forming tissues in vitro and their characterisation by TER or impedance measurements. Special emphasis is put on the novel cellZscope® system from nanoAnalytics which allows real-time impedance recordings, thus providing TER and CCL of vital cell cultures on ThinCert™ membranes. In principal these electrical parameters have been shown to correlate with a multitude of cellular features, such as intercellular connectivity and cell morphology and may be used to draw conclusions upon cell migration [3], probiotic activity [4] and changes in G-protein activity [5] and junctional tightness [6].

www.gbo.com/bioscience2 6

Figure 1: Formation of tight junctions between MDCK-II cells on a ThinCert™ permeable membrane support. MDCK-II cells have been cultivated on ThinCert™ cell culture inserts. After three days in vitro the cells were fixed on the membrane, cross-sectioned and stained with an antibody against ZO1. Immunoreactivity against this protein (arrow heads) indicates the presence of tight cell-cell junctions (nuclei counterstained with DAPI).

Figure 2: TER and impedance measurements with chopstick-type electrodes and the cellZscope® system.

A: Traditionally, chopstick-type electrodes (E1, E2) are used to measure the electric resistance of barrier-forming cell cultures (Bc) on ThinCert™ (Ti) permeable membrane supports (Pm). The ohmic electric resistances of the barrier forming cell layer (transendothelial or transepithelial electric resistance, TER), the cell culture medium in the upper and lower compartment (RMed), the permeable membrane support (RPm), as well as the electrode-medium interface (RE) contribute to the total electric resistance.

B: The cellZscope® system records the frequency-dependent impedance of an AC circuit with two wide electrodes (E1, E2) exhibiting a homogeneous electric field across a barrier forming cell culture and a ThinCert™ permeable membrane support. The mathematical model describing the impedance of this system includes the capacitance of the cell layer (CCL), a constant phase element (CPE) representing the electrodes, the ohmic electric resistances of the cell layer (TER), the permeable membrane support (RPm), and the cell culture media in the upper and lower compartments (RMed). Applying a best fit algorithm the cell layer related parameters TER and CCL may be calculated from the impedance data set.

f=1 Hz...100 kHz

Material and Methods

Material

All material used in this technical note is listed in table 1.

Table 1: Material and suppliers.

Item Supplier Cat.-No.

Alexa Fluor 488 goat anti-rabbit IgG

Invitrogen Corp. A-11008

BSA Sigma-Aldrich A7979

DRAQ5™ Biostatus Limited DR50050

EBSS Invitrogen Corp. 14155

EGTA Sigma-Aldrich 03777

EMEM Sigma-Aldrich M2779

FBS superior Biochrom AG S0615

L-Glutamine Biochrom AG K2083

MDCK-II ECACC 621027

Paraformaldehyde Sigma-Aldrich P6148

PBS Biochrom AG L1825

Pen/Strep Biochrom AG A2212

Aqua-Poly/Mount Polyscience 18606

ThinCert™ cell culture inserts, different membrane qualities

Greiner Bio-One GmbH 665 640, 665 641, 665 610, 662 630, 662 631, 662 638

Rabbit anti-ZO-1 Invitrogen Corp. 61-7300

TritonX100 Sigma-Aldrich T8787

Cell Culture

MDCK-II cells were grown in EMEM medium with Earle‘s Salts supplemented with 5% FBS, 2 mM L-Glutamine and 1% Pen/Strep. Cells were fed twice a week and split once a week. For the experiments cells were seeded on 12-well ThinCert™ cell culture inserts at a density of 5 × 105 cells × cm-2. For further cultivation and analysis inserts were placed in the cellZscope® system from nanoAnalytics.

Impedance (TER and CCL) measurements

The impedance of the cell cultures were measured automatically with the cellZscope® while the module holding the inserts remained in the incubator throughout the experiments (except for media exchange etc.) for maintaining physiological conditions. In case of comparative TER measurements with the EVOM hand-held device and STX2 chopstick-type electrodes (World Precision Instruments), the insert holding module or multiwell plates (Greiner Bio-One) had to be removed temporarily from the incubator and transferred to a flow bench. The chopstick-type electrodes were inserted in three different positions (120 deg turns) in each well and the readings averaged.

Immunocytochemical analysis

Cell layers were fixed at room temperature for 15 min in 4% Paraformaldehyde in PBS and then washed with PBS twice. For subsequent permeabilisation, cells were exposed to 0.5% TritonX100 in PBS for 10 min, followed by washing with PBS twice, exposure to 3% BSA in PBS for 20 min at room temperature and final washing with PBS three times. Fixed cell layers were incubated for 90 min at 37 °C with the primary antibody Rabbit anti-ZO-1 diluted 1:100 in PBS with 0.5% BSA, followed by washing in PBS three times, exposure to 3% BSA in PBS for 20 min at room temperature and final washing in PBS. Thereafter, cell layers were stained for 60 min at 37 °C with the secondary antibody Alexa Fluor 488 goat anti-rabbit IgG diluted 1:1000 in PBS with 0.5% BSA, followed by washing in PBS three times. Finally, cell layers were stained for 10 min at room temperature with DRAQ5™ diluted 1:1000 in PBS, followed by washing in PBS three times. Samples were mounted in Aqua-Poly/Mount on cover slides for subsequent analysis with a SPE confocal laser scanning microscope (Leica).

EGTA treatment

One cell culture served as a reference and was fixed and stained prior to EGTA treatment. All the other cell culture inserts were washed two times with Ca2+-free EBSS (Gibco) and further cultivated using serum free medium containing 0 (control), 1 or 4 mM EGTA. When TER was reduced by more than 50%, cell cultures of each experimental condition were fixed and subjected to immunocytochemical analysis. The EGTA containing cell culture medium in the remaining inserts was replaced by normal, Ca2+-containing medium and cell cultures were carried on.



Results

Real time monitoring of tight junction formation in the cell layer

MDCK-II cells were seeded onto 12-well ThinCert™ permeable membrane supports and TER and capacitance values were monitored over 140 hours using the cellZscope® system. The steep increase in TER observed as early as 5 hours after cell seeding (Fig. 3) marks the early onset of tight junction formation in the cell layer. After 10 hours, TER reached a maximum, indicating fully established tight junctions. The subsequent decrease in TER may be ascribed to an increasing number of cells and an increasing total perimeter of cell-cell contacts per surface area. Disturbances of the cell culture conditions such as the exchange of the cell culture medium and serum removal after 70 hours are well reflected in TER fluctuations (dashed line in Fig. 3). After 100 hours in culture, TER values reached a steady plateau between 70 and 120 Ω × cm². The onset of tight junction formation also correlated with decreasing CCL values.

In addition, the electric resistances of the ThinCert™ membranes alone have been determined using the cellZscope® system. Depending on the membrane characteristics, these values range from 25 to 75 Ω × cm² (Table 2).

Table 2: Electric resistance (RPm +RMed) of different ThinCert™ membranes.

Cat.-No.Membrane quality

Pore size in µm

RPm + RMed in Ω

665 640 translucent 0.4 26.7 ± 0.8

665 641 transparent 0.4 72.3 ± 1.3

665 610 transparent 1.0 33.3 ± 0.5

665 630 translucent 3.0 25.6 ± 0.9

665 631 transparent 3.0 28.4 ± 0.2

665 638 translucent 8.0 31.8 ± 2.0

Real-time monitoring of tight junction break-up

In another set of experiments, MDCK-II cells were cultivated on ThinCert™ cell culture inserts to confluence in order to form tight cell-cell junctions (Fig. 4B). TER and CCL were continuously recorded using the cellZscope® system. After 169.8 hours EGTA was added to the cell cultures, thus leading to a depletion of extracellular Ca2+ and thereby to disruption of the established tight junctions [7] (Fig. 4C). This process was well reflected in decreasing TER and increasing CCL. After removal of EGTA, tight junctions could be restored – TER and CCL values changed accordingly (Fig. 4A). The observed correlation of immunocytochemical findings and data deduced from impedance measurements strongly supports the concept of label-free analysis solely based on the electrical properties of the barrier-forming cell culture.

Comparison of TER measurements using chopstick-type electrodes and the cellZscope® system

Chopstick-type electrodes are still the most prevalent tool for TER measurements on cells cultivated in inserts. For comparison of the two approaches for TER measurement – the traditional chopstick-type electrodes and the novel cellZscope® – an additional set of experiments was performed. MDCK-II cells were cultivated on ThinCert™ membrane supports. One set of cell cultures was placed in a first cellZscope® system, which remained in the incubator throughout the experiment and was employed to continuously record TER values. A second cellZscope® system was loaded with a similar set of cell cultures. Again, the cellZscope® was used to automatically measure TER. However, at certain time points, the module holding the inserts was temporarily removed from the incubator and comparative TER measurements were performed with chopstick-type electrodes.

Figure 3: TER and CCL recordings on MDCK-II cells cultivated on ThinCert™ cell culture inserts with different pore sizes.Increasing transepithelial electric resistance values (TER) correlate with a decreasing capacitance (CCL) of the cell layer. After 60 hours the TER values level out at 100 -160 Ω × cm², thus indicating confluence and formation of a tight cell layer. The dashed line indicates the time point of medium exchange.

Interestingly, the TER plateau reached after long-term cultivation was significantly higher in the experimental setup that was not disturbed by manual chopstick measurements outside the incubator. This finding is indicative of the relatively strong interference of this classical measurement approach with cell culture conditions. The more cells are cultivated in a continuous and undisturbed manner and culture conditions are kept constant, the more cellular parameters may reach a stable level. The cellZscope® system supports this requirement with its non-invasive operating principle.


Figure 5: TER measurements on MDCK-II cells cultivated on ThinCert™ cell culture inserts using chopstick-type electrodes and the cellZscope® system.MDCK-II cells cultivated on ThinCert™ cell culture inserts develop higher TER values when cultivated in the undisturbed cellZscope® system as compared to cultivation in the cellZscope® with repeated disturbance by manual measurements with chopstick-type electrodes. TER values obtained with chopstick-type electrodes are slightly higher, but correlative with values acquired with the cellZscope system®.

Data points taken with the chopstick-type electrodes give a rough indication of the typical time-dependent development of TER during cell differentiation and tight junction formation (Fig. 5). TER values determined with the chopstick-type electrodes correlated with those continuously acquired with the cellZscope® system, but were found to be slightly higher, which may be explained by inhomogeneous electrical fields between the chopstick-type electrodes, resulting in a systematic overestimation of TER values.

Figure 4: Break-up of tight junctions correlates with decreasing TER and increasing CCL values.

A: TER and CCL of a MDCK-II culture have been recorded continuously using the cellZsope® system. After 169.8 hours in vitro, EGTA has been added to the cell culture medium at varying concentrations (0 mM, 1 mM, 2 mM) and tight junctions have been broken up. Tight junction break-up correlates with a severe drop of the transepithelial electric resistance (TER) and an increase of the capacitance of the cell layer (CCL).

B, C: Immunocytochemistry using an anti-ZO1 primary antibody confirms the presence of tight junctions in the cell layer (B) and their disappearance after treatment with 2 mM EGTA (C). Nuclei were counterstained with DRAQ5™. Images have been acquired with a confocal microscope. Note that the image in C represents the confocal plane with most remaining ZO1 immunogenicity, thus making nuclei appear smaller than in B.

References

1 Wegener J, Abrams D, Willenbrink W, Galla HJ , Janshoff A. Automated

multi-well device to measure transepithelial electrical restistances under

physiological conditions. BioTechniques 2004 Oct; 37(4):590-97.

2 nanoAnalytics GmbH. Monitoring Barrier Properties of MDCK Cell

Layers During Depletion of Cell Cholesterol by Methyl-β-Cyclodextrin.

www.nanoanalytics.com.

3 Ludwig T, Ossig R, Graessel S, Wilhelmi M, Oberleithner H, Schneider SW.

The electrical resistance breakdown assay determines the role of proteinases

in tumor cell invasion. Am J Physiol Renal Physiol. 2002 Aug;283(2):F319-27.

4 Klingberg TD, Pedersen MH, Cencic A, Budde BB. Application of

measurements of transepithelial electrical resistance of intestinal epithelial

cell monolayers to evaluate probiotic activity. Appl Environ Microbiol. 2005

Nov;71(11):7528-30.

5 McGuinness RP, Proctor JM, Gallant DL, van Staden CJ, Ly JT, Tang FL,

Lee PH. Enhanced selectivity screening of GPCR ligands using a label-free

cell based assay technology. Comb Chem High Throughput Screen. 2009

Sep;12(8):812-23.

6 Wedel-Parlow M, Wölte P, Galla HJ. Regulation of major efflux transporters

under inflammatory conditions at the blood-brain barrier in vitro.

J. Neurochem. 2009 Jun; 111:111-18.

7 Rothen-Rutishauser B, Riesen FK, Braun A, Günthert M, Wunderli-Allenspach

H. Dynamics of Tight and Adherens Junctions Under EGTA Treatment.

J Membrane Biol. 2002 Apr; 188: 151.

8 Letschert K, Komatsu M, Hummel-Eisenbeiss J, Keppler D. Vectorial

transport of the peptide CCK-8 by double-transfected MDCKII cells stably

expressing the organic anion transporter OATP1B3 (OATP8) and the export

pump ABCC2. J Pharmacol Exp Ther. 2005 May;313(2):549-56.

AcknowledgementWe thank Dr. Boris Anczykowski from nanoAnalytics GmbH for supporting the

experiments with the cellZcope® system.

We thank Kathrin Hardes, Kristina Riehemann, Vanessa Heitmann, Martin Kahms

and Jana Hüve from the Westfälische Wilhelms-University of Münster for carrying

out all cell culture experiments and immunocytochemical analyses.


Conclusion

ThinCert™ cell culture inserts allow the restoration of in vivo-like growth conditions in vitro and hence the reconstruction of epithelia and endothelia from individual cells. The cultivated cell layers develop tissue-specific features, such as tight junctions, cellular polarity and barrier function, and may be used to study tissue-specific phenomena, such as vectorial transport in vitro [8]. The transendothelial and -epithelial electric resistance provides a helpful tool to assess the properties of the cultivated cells in a non-invasive manner. Novel devices, such as the cellZscope® system from nanoAnalytics, further extend the possibilities of cell-based research with ThinCert™ cell culture inserts by enabling continuous real-time analyses, providing additional information about the capacitance of the cultivated cell layers and increasing assay throughput (for comparison of the classical and novel approach see Table 3).In the future, impedance-based analyses may be used to non-invasively analyse any cellular property that is correlated to altered cellular conductivity or cell shape. Such putative applications may include: studies on cell migration, alterations in membrane composition, changes of cell-cell contacts as well as the examination of signalling cascades leading to rearrangements of the cytoskeleton and changes of the overall cell shape and morphology.

Table 3: Comparison of classical and novel ways of analysis of cell cultures based on electrical properties.

Instrument EVOM + STX2 electrodes

cellZscope®

Suitability for ThinCert™ cell culture inserts

yes yes

Measured parameter of cell layer

TER TER and capacity (impedance)

Related cellular features

n conductivity of the cell layer

n formation of tight cell-cell contacts

n conductivity of the cell layer

n formation of tight cell-cell contacts

n formation of cellular protrusions

n changes in cell shape and morphology

Application field: n determination of barrier function of a cell culture

n end point analysisn single or low

throughput analysis

n determination of barrier function of a cell culture

n complex correlation of electrical and cell-biological properties of cell cultures

n real-time analysisn medium throughput

analysis

Acquisition cost low medium

Germany (Main office): Greiner Bio-One GmbH, [email protected] l Austria: Greiner Bio-One GmbH, [email protected]: Greiner Bio-One BVBA/SPRL, [email protected] l Brazil: Greiner Bio-One Brasil, [email protected]: Greiner Bio-One GmbH, [email protected] l France: Greiner Bio-One SAS, [email protected] Japan: Greiner Bio-One Co. Ltd., [email protected] l Netherlands: Greiner Bio-One B.V., [email protected] UK: Greiner Bio-One Ltd., [email protected] l USA: Greiner Bio-One North America Inc., [email protected]

Revision June 2010 - F073 037

Documents

Tissue Culture Inserts for Multiwell Plates - file.yizimg.comfile.yizimg.com/472786/2013071112040270.pdf · Greiner Bio-One offers tissue culture inserts for 6, 12 und 24 well multiwell