Use of new technology of Oxygen controlled disposable ... · PDF fileUse of new technology of Oxygen controlled disposable bioreactors for cell growth ... of Oxygen controlled disposable

2012

Celartia V.Russo & E. Barbera-Guillem

Use of new technology of Oxygen controlled disposable bioreactors for cell growth and cellular therapy applications Ducted Respiratory Chambers (Petaka G3) have been used in many laboratories all over the world with very successful results proving that cells can be cultured in physiologic level of Oxygen (50 mmHg) whilst in normal external atmosphere with 21% oxygen. Classic devices can only achieve this by incubating cultures in hypoxia chambers with 5% Oxygen (which still gives no indication of the actual DO in the media).

Celartias’ White Paper Nov. 2012 ___________________________________________________________________

2

Use of new technology of Oxygen controlled disposable bioreactors for cell growth and cellular therapy applications

Developments in stem cell culture for regenerative medicine and diverse cell lines for drug discovery are creating demands for cell culture under true physiologic conditions mimicking the atmosphere within living beings with the intent of avoiding misleading results as produced in classic cell culture systems in hyperoxia. While objectives in days gone by, were simply to adequately maintain cells alive and genetically stable, to avoiding contamination, and to grow cells quickly as possible in large numbers; investigators, medical doctors and industry now recognize the subtle, but dangerous & far reaching effects of unnatural cellular environments. Atmospheric oxygen concentrations of classic open in vitro cultures are far higher than those experienced by in vivo cells, and this difference has far reaching consequences. Room air oxygen levels in cultures increase oxidative metabolism, mitochondrial activity, and forced cell proliferation, inhibiting other cell functions, including exportable protein synthesis. These effects are translated in erroneous in vitro estimations of sensitivity of tumor cells and normal cells to drugs in development and radiation, reduced protein production in pharmaceutical industry and distorted data in signal transduction and cellular activation/inhibition in costly and important cell biology research. In traditional plastic labware, namely, flasks, bottles and plates, cells grow as a monolayer or in 3D clusters below a shallow layer of media which in turn is directly exposed to air containing 21% oxygen, which is rapidly and constantly dissolving in the media up to saturation limits, whilst in true in vivo cellular conditions, oxygen levels are much lower. Without complex active interventions in classic incubators, cells are exposed to free radical formation inducing atmosphere, prevalent oxidative metabolism and mutagenesis. Moreover, classic cell cultures are buffered with bicarbonate that need a permanent source of CO2 for maintaining a neutral balance. CO2 produced by normal cellular molecular combustion, rapidly escapes from the media to the atmosphere if the CO2 in the atmosphere is not artificially raised high enough, raising the pH to deadly levels. Open atmosphere contains almost no carbon dioxide, so supplemental industrial CO2 is supplied in canisters and injected in the incubator atmosphere with regulating devices. Furthermore, normal cell cultures are maintained at 37⁰C which, as a side effect, facilitates a rapid dehydration of the cultures, unbalancing the osmolarity and the molecular concentrations


3

of the nutrients. This serves to avoid dehydration of the culture, by creating a saturated water environment inside the incubator (water pan). Exclusion of oxygen at physiologic levels is particularly problematic, requiring sealed incubators, nitrogen flushing, and repeated attention to preventing the ingress of omnipresent atmospheric oxygen during regular & constant incubator door opening for access to the cultures. The independence from the necessity of gas regulation in the DRC ensures that cell cultures & gene expression are constant & repeatable having no external influences even in the face of regular accessing of shared incubators. A plethora of complex hypoxia chambers and glove boxes have been developed as active interventions to bring oxygen, carbon dioxide and humidity levels in line with true physiologic conditions, but despite this, wide fluctuations and failure of these active interventions are a constant danger and a problem issue for compliance with the terms of GMP protocols .

Instead of multiple active attempts to replicate natural conditions, Celartia took a new direction, employing engineered devices, based on Ducted Respiratory Chamber Systems (DRC). We created the Petaka® G3, which instead of attempting to impose “normal” gas conditions on the cells through active incubator control; it passively allows cells to maintain their own, truly normal oxygen, carbon dioxide, and humidity levels. These levels are maintained automatically and independently from the laboratory or incubator conditions. Because the DRC devices act passively, they are inherently far simpler, more reliable, and less expensive than traditional methods of active gas control.

A New Bioreactor Fosters Naturally Produced Physiologic Conditions

While the passively controlled gas environments within the DRC are far simpler than the active interventions required by today’s incubators, they are actually far more dependable, as they are driven exclusively by natural laws. The Petaka® G3 DRC design

is seen schematically in Fig. 1.

Natural Laws Drive DRC Function

The cell culture chamber is isolated on the injection side from the atmosphere by a self-sealing silicone injection port that allows the closed introduction of media and cells, including most eukaryotic cells types, small early stage embryos, tissue fragments and even needle biopsies.

Figure 1. Schematic of Ducted Respiratory Bioreactor (DRC).


4

On the venting side, the culture chamber is partially isolated from the atmosphere by an engineered, semi-closed respiratory duct having two major portions. Closest to the reaction chamber, a series of small chambers which act as water vapor condensers blocking evaporation, preventing alterations in media osmolarity, even at 37⁰ C for 10 to 20 days at 10% RH. The chambers also act as a capillarity breaker to prevent media flow into the second portion: the respiratory duct that controls internal gas levels. The length and cross-section of the respiratory duct is purposely engineered to partially restrict the diffusion of oxygen from the high levels of ambient air to create lower, physiologic levels of dissolved oxygen in the reaction chamber. In accordance to Fick’s Law, as oxygen is consumed inside the culture chamber, decreasing the partial pressure of oxygen in the media, oxygen diffuses in from the outside atmospheric (higher) partial pressure, through the respiratory duct, to the lower partial pressure inside. Diffusion is proportional to the concentration

gradient, as regulated by the engineered design of the respiratory duct, and occurs entirely spontaneously and without any manual intervention whatsoever. These physical features of the Petaka® G3 are seen labeled in Fig. 2. Figure 2. Petaka®G3 DRC. (1) cell culture chamber;(2) injection port; (3) respiratory duct; (4) 0.2μ filter; (5) water vapor condensers and capillary breakers; (6) unique barcode. The DRC is shown upright in a silicone stand (7).

At the same time, the respiratory duct partially retains carbon dioxide from cellular metabolism to maintain a

physiologically normal mild acidosis to balance pH. All gas exchange with the outside environment occurs via a 0.2 micron filtered vent, preserving the internal sterility of the device but allowing exchange of gas diffusion and flow to prevent pressurization issues when filling and emptying the bioreactors.

Therefore, cell culture in DRC’s/Petaka G3 does not require supplemental oxygen-nitrogen balancing, CO2 and humidity sources, eliminating the entire panoply of gas tanks, regulators, sensors, microprocessors and water pans. This creates a double benefit: not only are cells cultured in more normal physiologic conditions, but the


5

mechanics, logistics, risks and costs of cell culture are greatly simplified and reduced. Because the gas environment is automatically maintained in Petaka G3, cultures can be incubated in virtually any kind of chamber that provides heat, … and nothing else.

An additional drawback to culturing cells in incubators with regulated oxygen and using the so called hypoxia chambers is the absolute lack of accurate readings with respect to how much oxygen is dissolved in the cell culture media microenvironment. Setting the oxygen dial to a given level of O2 concentration is by no means, an accurate guarantee of the actual amount of dissolved oxygen in the media, as it is dependent on many factors including gradients derived from the depth of the media layer in flasks and dishes, the changing viscosity of the media, the changes in the diffusion coefficient of the oxygen associated to the progressive changes of the media molecular constants, and more. So setting the O2 dial to 5% is only stating, in the best case scenario, that the atmosphere where the flasks are incubated only contain 5% oxygen but it is not saying what level of DO the cells are living in. Petaka G3-OS has an integrated internal oxygen sensor that can be probed any time during the life of the culture, delivering readings of the actual amount of DO in the media in a non-invasive manner without disturbing the cell culture progression or the oxygen diffusion rates.

Internal Dissolved Oxygen Sensor. An accurate tool for research and GMP.

Fig. 3 Direct reading of the Oxygen sensor included in Petaka G3-OS

The sensor is strategically placed at the level of the cell monolayer at a point of the cell culture surface equidistant of all oxygen diffusion spots, avoiding gradient influences in the readings, and easy to read with the external light probe (Fig.3)

Many genes promoting cellular differentiation and expression are down-regulated at atmospheric oxygen tensions. Naturally induced oxygen levels and cell growth in the DRC are seen in Fig. 4. In different experiments, series of DRC bioreactors were seeded with adult differentiated cell lines, 106 million cells per DRC in DMEM or RPMI 1640 media, and incubated in room air at 37.5OC. At time zero (left, Fig. 4) seed cells and media recently transferred from regular bottles exposed to room air O2 (DO~120 mm

Naturally Produced Conditions: First for Proliferation, Then for Differentiation


6

Hg) are injected into the DRC. Normal tissue O2 levels (5-50 mm Hg).

Figure 4. Self-Regulating Gas Management in the DRC. Cells consume O2, with restricted O2 ingress, first causing a proliferative cell phase to evolve into a second differentiating cell phase for protein production and gene expression.

Cells proliferate, consuming O2, causing the O2 level within the DRC to begin to fall, but restricted diffusion through the respiratory duct only partially replaces the O2. At about 5 x106 cell count, internal O2 levels enter the upper limits of physiologically normal tissues (~ 50 mm Hg). At about 8 x 106 cell count, O2 diffusion through the respiratory duct approaches compensatory levels for O2 consumption. At that level, cells enter a differentiation phase, characterized by a leveling of the doubling rate, expanding protein expression and normal gene expression. Because the cells control their own gas environment, there is little effect from outside gas conditions, and the DRC is effective in atmospheres from 500 meters below sea level up to elevations of 4,000 meters, and in relative humidity levels between 10% and 100%. The essential resulting difference between cell harvesting in classic devices & with DRC is that with flasks, having cultured in 5% CO2, upon opening the incubator door to collect the cultures, the cells are immediately exposed to 21% oxygen & therefore exposed to hyperoxic conditions during the harvesting process resulting in only


7

estimated readings. The closed system of the DRC allows cells to be detached & harvested still well within the recognized physiological normoxia band levels ensuring that any analysis will accurate & relevant.

Petaka G3 changes the paradigm of cell culture, replacing nearly a century of active attempts of human intervention to manipulate gas exchange, with a self-regulating, design driven by natural laws of gas diffusion to create dependable, and truly physiologic, dissolved gas environment in the liquid habitat of the cells in vitro, quasi identical to the in vivo conditions.

Fitting the demands of stem cell culture

Physiologic cell metabolism is achieved in DRC bioreactors incubated in unaltered open atmosphere. Petaka, the genuine DRC bioreactor, has been used for years for cell lines culture with great results and clear benefits demonstrating that oxygen control is essential for cell biology research and cell based protein synthesis if the results are to in any way mimic in-vivo.

Figure. 5. Evaluation of the maximal respiration capacity of CHO cells cultured in 21% oxygen and under controlled dissolved oxygen in Petaka (35 mmHg oxygen partial pressure in the media)

For example CHO cells cultivated under oxygen control live longer after cell confluent growth. In a study made in 2011 by Dr. Rachel Legmann (Seahorse Bioscience, Boston, Massachusetts, USA ) using XF instruments and technology it was demonstrated that the Basal Respiration Capacity and Maximal Respiration capacity (FCCP) of cells do not change under oxygen control in PetakaG3 (Fig. 5), however the relative protein production under oxygen control experimented 60% increase under oxygen control compared to cultures in flasks and dishes. More experiments in the same laboratories by Dr. R. Legmann 1, 2

1

culturing a C3H mouse myoblast cell line (C2C12, Yaffe and Saxel, 1977) showed that the final cell yield

www.seahorsebio.com

http://www.seahorsebio.com/�


8

in Petaka was at least 50% higher in PetakaG3, the protein synthesis 40% higher maintaining a Max. Respiration capacity below 50% of this observer in hyperoxic conditions in T flasks cultures (Fig 6)

Figure 6.- Behavior of myoblast cells with differentiation capacity cultured in physiologic oxygen (blue and magenta bars) versus cultures in flasks and dishes exposed to 21% atmospheric Oxygen (red bars)

Experiments developed by Sofie Claerhout ( at The University of Texas, M.D. Anderson Cancer Center,Houston, Texas) demonstrate the utility of Petaka in the investigation of tumor cell apoptosis and autophagy under physiologic oxygen3

Different variants of the Jurkat cell line cultured either in high oxygen in flasks or in physiologic oxygen in Petaka were tested for expression of a broad panel of characteristic CDs by flow cytometry including: CD1a, CD3, CD5, CD10, CD14, CD16, CD19, CD26, CD38, CD39, CD56, CD57, CD69, CD73, CD138, CD157, PC-1, TfR-2, HLA-I, HLA-II. Qualitative analysis showed that the classes of CD are identical in both oxygen environments and quantitative differences were shown (Dr.F. Malavasi. Laboratory of Immunogenetics, University of Torino. Medical School. Torino. Italy)

Phenotype and cluster of differentiation (CD) are maintained in physiologic Oxygen availability provided by DRC bioreactors incubated at open atmosphere

2 www.seahorsebio.com/resources/pdfs/brochure-XFe-2012.pdf 3 Sofie Claerhout et al. Abortive Autophagy Induces Endoplasmic Reticulum Stress and Cell Death in Cancer Cells. PLoS One. 2012; 7(6): e39400. Published online 2012 June 26. doi: 10.1371/journal.pone.0039400

http://www.seahorsebio.com/resources/pdfs/brochure-XFe-2012.pdf�


9

Avian Mesenchymal Stem Cell

Physiologic stem cell growth and differentiation is achieved in DRC bioreactors incubated at open atmosphere.

Differentiation is achievable in Petaka at low levels of Oxygen as demonstrated in primary cultures of Avian Mesenchymal Stem Cells (AvMSC), which grow at 40 to 50 mmHg of O2 tension to be confluent with undifferentiated characteristics. Grown on a matrix of CellGrip (NEURO-ZONE, Milano, Italy) When cells reached the confluent point the level of DO is 15 mmHg, and then a simple reduction of temperature to 24OC (mild hypothermia) induces differentiation into fibroblasts and primitive multi-locular adipocytes, maintaining the potential to reverse into the more primitive status (Fig 7) a few times raising the temperature back to 37OC for 8 hours. Fig. 7 - Development of avian stem cells in Petaka G3 on a matrix of CellGrip. A) Initial growth of 3D clusters of primary stem cells. B) Growth and migration of differentiating cells from the primary clusters. C) Confluent monolayer of stem cells and differentiating cells. D) Spontaneous differentiation of these cells in primitive adipocytes as a response to mild hypothermia (24OC). V. Russo. Development of avian stem cell cultures in Petaka G3-LOT. Photos: V. Russo. Celartia, July 2011

In the same line these results could be related with the mechanisms that promote fat differentiation under hypoxia of the mammalian myoblasts C2C12.4

In other studies with mouse derived MSC, after sustained confluent growth over 72 hours in Petaka the cells adopted a polygonal star like shape in which M.Burgal (Prince Felipe Research Center, Valencia, Spain) was able to identify a kind of epithelial-like

Mouse Mesenchymal Stem Cells

4 Itoigawa Y, Kishimoto KN, Okuno H, Sano H, Kaneko K, Itoi E. Hypoxia induces adipogenic differentitation of myoblastic cell lines. Biochem Biophys Res Commun. 2010 Sep 3;399(4):721-6.


10

differentiation with non-organized deposits of glyal fibrilar acidic protein expression in the cytoplasm after 3 day of spontaneous differentiation in confluent monolayer at 20 mmHg of oxygen partial pressure, by in-situ immune-fluorescence staining using anti-GFAP and a Leica confocal microscope (Fig 8) Figure 8. Immuno-fluorescence microscopy of a mouse MSC in differentiation progression. Culture under 20 mmHg of O2 partial pressure. Red fluorescence positive staining of GFAP.

Cord Blood-Derived Human Mesenchymal Stem CellsHuman Mesenchymal Stem Cells

(hMSC) were cultured in Petaka without any matrix and maintained in MSC-GroTM low serum media yielded identical

results based on growth,

Fig. 9 hMSC 3 hours after seeding in Petaka G3 with low serum media and without matrix (Left) and 96 h later (Right). Photos: Jim Musick. Vitro-Biopharma. September, 2012.

doubling time and differentiation, as culturing cells in dishes coated with a matrix in induced hypoxia conditions.

(Dr J. Musick. VitroBiopharma, Colorado. USA.2012 ) (Fig.9). These cells after being cultured in Petaka and maintained at room temperature for 8 days were able to grow immediately with minimal cell losses. When these cells were exposed to mild hypothermia they adopted more fibroblastic shape but never showed any other signs of differentiation (Fig.10).

http://vitrobiopharma.com/vitro_products/catalog-number-sc00a1-cord-blood-derived-human-mesenchymal-stem-cells/�


11

Fig. 10 hMSC cultured for 5 days at 37OC in Petaka G3 with low serum media and without matrix then maintained in Petaka G3 for 8 days at RT and reincubated at 37OC for 3 hours (Left. Edge region. Right. Midregion). Photos: Jim Musick. Vitro-Biopharma. September, 2012.

Cord Blood-Derived Human Mesenchymal Stem Cells on CellGrip Matrix

However the same cells when transported frozen, showed a viability of only 68% 5 minutes after the thawing. Figure 11. hMSC growing in Petaka G3 coated with CellGrip. Photo Celartia 2012. When the survival 7.5 x105cells from that sample were seeded in Petaka with CellGrip matrix cell proliferation was evident in 8 hours with cells developing monolayers (Fig 11). On this matrix, cells adopt a spread fibroblastic phenotype similar to endothelial cells (Fig. 11) FIgure 12. Human MSC cultured in Petaka coated with CellGrip at a level of DO partial pressure of 15 mmHg for 24 h. Photo N. Abasolo. Celartia. October, 2012

http://vitrobiopharma.com/vitro_products/catalog-number-sc00a1-cord-blood-derived-human-mesenchymal-stem-cells/�


12

After the cell monolayer was confluent the O2 internal partial pressure reaches 15 mmHg and cells start showing differentiation patterns. In this example cells acquire a specific shape with cytoplasmic stripes in geometrical patterns similar to the myofibril bundles of rhabdo myoblasts (not molecular identifications were performed, only morphologic evaluation) (Fig. 12)

iPS Cells has been also cultured in Petaka G3 with specific matrixes with excellent results, in the Lerner Research Institute at the Cleveland Clinic, Department of Stem Cell Biology and Regenerative Medicine. The goal of this team, under the leadership of Dr. Jan Jensen, is the differentiation of these cells into endocrine pancreatic cells.

Induced pluripotent stem cells (iPS cells)

Harvesting anchorage dependent cells necessitate a phase of cell release or detachment from the substrate which traditionally implies incubation of the culture in media containing active proteolytic enzymes able to break the molecular links between the cells and surface of the culture device. This treatment can be traumatic for the cells, and should be well executed in order to minimize cell stress or damage to the general functions of the cells. However there are several inherent flaws to this procedure:

Enzyme free cell detachment An additional improvement incorporated to Petaka G3.

Figure 13. The Cell Flipper tows the internal gliding devices of Petaka G3-ET generating vortexes capable of detaching the cells. Firstly, it is an “all or nothing” method. The enzyme solution is applied to the entire cell culture device after removing the media and washing the residual traces of serum, when this is used. All cells are detached and the device is not suitable for cell regeneration. Secondly, the protocol is quite traumatic and time consuming because after all cells are detached, these should be washed again to eliminate all traces of enzyme. This includes several passages to tubes and centrifugations.


13

Thirdly, there always exists the possibility of having altered the cell molecules on the cell surface involved in many critical intercellular relations and functional responses, which are particularly critical in the case of stem cells5,6

Therefore, notably for stem cell harvesting the specialists recommend the use of mechanical rather than chemical means to detach the cells such as scrapers Pasteur pipettes, etc.

.

A closed bioreactor like Petaka does not allow for external or invasive mechanical interventions on the cells; however, there exists special version of Petaka G3 called Petaka G3-ET (Easy Transfer) which contains several paramagnetic micro devices built inside which are designed to glide over the cells when mobilized by an external magnetic field at a specific speed. These flying micro devices generate horizontal vortexes, parallel to the cell culture surface resulting in cell detachment without the need of any enzyme or scraping. The mobile magnetic fields are generated by a novel device called the Cell Flipper (Fig 13). Figure 14.- 3D cellular aggregates produce after enzyme free cell detachment. CHO cells.

Using such a system, cells are detached in seconds and also a limited partial cell release is possible allowing just sampling the culture or seeding in other devices and maintaining the donor culture unaltered. Detachments without enzymes maintain the cell surface molecular atmosphere intact allowing cell aggregation. Cellular aggregates made with this method are pure without intercellular matrix except the molecules produced by the cells themselves and can be isolated and cultured as 3D pseudo tissues Fig 14.

Cell detachment by internal vortex in Petaka G3-ET, when cultured cells are about 80% confluent, deliver both single cells and cell aggregates in proportions related to the time of exposure to the flipper action (see table 1).

5 Jonathan S. et al. Draper Surface antigens of human embryonic stem cells: changes upon differentiation in culture. Journal of Anatomy. 249–258, March 2002

6 Manas K. Majumdar er al. Characterization and Functionality of Cell Surface Molecules on Human Mesenchymal Stem Cells Journal of Biomedical Science 2003;10:228-241


14

Figure 15.- Cell attachment and growth progression in Petaka G3, 4h after passage by enzyme free cell detachment with Cell Flipper. Only when cultures are overgrown the detachment can be hard and may generate significant level of cell death. However, detachments of 80% confluent healthy cultures produce negligible proportion of unviable cells and after transferring, the cells attach and grow in a few hours (Fig.15)

Table 1. Yield of enzyme free cell detachment with Petaka G3-ET and Cell Flipper

CONCLUSIONS Ducted Respiratory Chambers (Petaka G3) have been used in many laboratories all over the world with very successful results proving that:

1. Cells can be cultured in physiologic level of Oxygen (50 mmHg) whilst in normal external atmosphere with 21% oxygen. Classic devices can only achieve this by incubating cultures in hypoxia chambers with 5% Oxygen (which still gives no indication of the actual DO in the media).

2. In physiologic level of Oxygen, many different cell types maintain the signature molecular markers. Changes have been observed only at quantitative levels, proving that culturing in hyperoxia may deliver important artifact data.

3. In physiologic level of Oxygen mesenchymal stem cells and iPS cells grow and differentiate more efficiently even reducing dependence on extra cellular matrices.

Flipper exposure seconds

% monolayer released

% of aggregates

% Cell Viability

5 15 70 99 10 40 60 98 15 55 45 98 30 90 10 90 45 95 5 89 60 98 3 87


15

4. Petaka G3, the genuine Ducted Respiratory Chamber, is a robust and reliable instrument for culturing cells in Respiratory Physiologic conditions, mimicking the natural environment of the tissues.

5. Petaka G3 is ideal for stem cell culture and experimentation. 6. Enzyme free cell detachment with internal vortexes, in unchanged Oxygen levels,

provides single suspended cells and 3D cell aggregates in time dependent proportions.

Emilio Barbera-Guillem MD, PhD CSO Celartia Ltd.

Copyright Celartia Ltd. 2012

Documents

Use of new technology of Oxygen controlled disposable ... · PDF fileUse of new technology of Oxygen controlled disposable bioreactors for cell growth ... of Oxygen controlled disposable