4. Three dimensional growth of animal cells on PDLLA scaffolds

4.1 Introduction

4.2 Materials and Methods

4.3 Results

4.4 Discussion

4.5 Conclusions

4.6 References

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4.1 Introduction

An emerging philosophy in tissue engineering is that the scaffold fabricated from

synthetic biomaterial should establish key interactions with the seeded cells and coax the

innate power of cells to organize into three dimensional tissue structures (1 ). Suitability

of scaffold for cell growth depends on many factors. Complexity of interactions of

biomaterial with the living cells need proper evaluation for the efficient use of

biomaterial derived scaffolds. Proper attachment of cells onto the biomaterial surface and

reduced toxicity of the biomaterial are the two main factors responsible for cell growth.

In order to achieve this, scaffolds are generally treated to improve wettability and

adhesion properties. Generally scaffolds are treated with serum which contains many

attachment factors beneficial for cell attachment and growth. As PLA/PLGA polymers

are hydrophobic and have very little cell adhesive properties, they are pretreated in

different ways to promote cell growth (2, 3). In the present case, preparation of the

scaffold was carried out in steps as detailed in the earlier chapter. First, surfactant coated

large porous PDLLA particles were prepared. Secondly, polymeric particles were fused

in presence of ethanol to form higher order structures. It is thus essential to see if the

process of scaffold fabrication has any deleterious effect on cell growth.

In this chapter, the PDLLA-CTAB scaffolds were primarily evaluated for the three

dimensional growth of cancer cells. These scaffolds were made to undergo a pretreatment

protocol to make them suitable for cell growth. On seeding scaffold with cells, the cells

attached, spread and proliferated into three dimensional structures. Cancer cell lines

grown and perpetuated on tissue culture plates have been extensively used by biologists

to study various aspects for cancer biology. Pioneering work by researchers like Mina

Bissel pointed towards the various limitations of two dimensional cultures and

established the relevance of three dimensional cell cultures especially in relation to

cancer biology (4). The characteristic features of the tumor microenvironment like the

chronic hypoxic conditions which promotes drug resistance and tumor cell invasion are

best studied in a three dimensional context (5, 6). It is very essential to standardize three

dimensional cancer models to study tumor biology and use it as a model for drug testing.

The topology and surface characteristics of the scaffold formulated by the self-assembly

71

of polylactide particles proved to be suitable in promoting the attachment, proliferation

and growth of the cancer cell lines into three dimensional structures. Cancer cells grown

on polymeric scaffolds can be a viable alternative to monolayer cultures for evaluation of

anti cancer drugs (7, 8). This can be used as an in vitro model for understanding various

aspects of tumor biology (9-12). Breast cancer and melanoma cell lines were used for

three dimensional growth on scaffolds and as a model for drug testing. One of the

deadliest features of cancer is its ability to invade surrounding tissue and metastasize to

distant sites. It is necessary to develop drugs that can fight and control the invasive nature

of tumors. Evaluation and screening of anti-metastatic drugs requires a suitable in vitro

models and monolayer cultures are not adequate to this task. Three dimensional cultures

of the cancer cells were kept on collagen gel to study tumor cell migration so as to

develop a model for evaluation of potential anti-metastatic agents. The main objective

was to evaluate the suitability of scaffold for cell growth and use them as an in vitro

model to understand cancer biology with respect to drug sensitivity.

4.2 Materials and Methods

4.2.1 Materials PDLLA (45 kDa) was from Durect Corporation (Pelham, USA).

Cetyltrimethyl ammonium bromide (CTAB) was procured from Amresco chemicals,

USA. Fetal calf serum (FCS) and Roswell Park Memorial Institute (RPMI-1640) medium

were from invitrogen, USA. 3D collagen kit was from Millipore, USA. DAPI and CFSE

fluorescent dyes were from Molecular probes, USA. LPS and PMA were from sigma

chemicals, USA.

4.2.2 Preparation of PDLLA-CTAB scaffolds Preparation of scaffolds from PDLLA­

CTAB particles has been described in detail in the chapter 3. For the growth of cancer

cells, 10 mg ofthe particles were weighed and spread out in a sterile Petri plate inside the

laminar flow hood and wetted with ethanol. The particles fused to form a membrane

which was washed with sterile water. The scaffolds were made to undergo a pretreatment

protocol to make them suitable for cell attachment and growth. Scaffolds of different

sizes were made by spreading the particles in Petri dishes of different sizes and fusing

them with alcohol.

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4.2.3 Surface characterization of PDLLA-CTAB scaffold for cell growth The scaffold

surface was visualized by scanning electron microscope (SEM)-model EVO 50 (Zeiss,

Germany). The images were taken after coating the particle surface with gold over an

aluminium stub. Atomic Force Microscopy (AFM) of the surfactant coated particle

surface after ethanol treatment was carried out to study surface topology using

Nanoscope (Veeco, USA). The samples were deposited onto mica supports or glass slide

and images taken. Amplitude-distance curves were used to optimize resolution and

contrast in semi-contact mode of atomic force microscope. Estimation of CTAB

concentration desorbed from the PDLLA-CTAB during alcohol and sterile PBS washes

were estimated using colorimetric method as described in chapter 3.

4.2.4 Pre-treatment of PDLLA-CTAB scaffold for cell growth PDLLA is a

hydrophobic polymer and the surface has to be made more biologically compatible for

the cells to initially attach and proliferate into three dimensional structures. In order to

make the PLA membrane based scaffold suitable for cell growth an elaborate pre

treatment procedure was developed. The pre treatment procedure has 4 steps.

Step 1: Fusion ofPDLLA-CTAB particles in alcohol to form scaffolds

Step 2: Treatment of scaffold with 100 % alcohol for three hours

Step 3: Thorough washing of the scaffolds using sterile PBS

Step 4: Treatment of scaffold with 15 % fetal calf serum for 12 hours

4.2.5 Culture of animal cells on polymeric scaffolds The average size of PDLLA-CTAB

scaffold used was about 0.2 sq em which was formed using 1 mg of PDLLA-CTAB

particles. They are easy to handle and can be transferred to culture wells with pipette. After

overnight serum treatment, the scaffolds were transferred to six well plates and were

incubated with 20 fll of RPMI complete media containing 0.1 million MCF -7 cells or other

cell lines for 50 minutes for cell attachment. The scaffolds were cultured in 1.5 ml of

complete medium in a C02 incubator and medium was changed on alternate days. B 16

melanoma cells were also grown on the treated scaffold as described for MCF -7 cells.

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Sigma cell dissociation buffer was used to dissociate the cells from the scaffold at different

time points to check for cell number and viability was assessed by trypan blue staining.

Three dimensional growth of cells on the scaffold was visualized by optical microscope.

DAPI (4, 6-diamidino-2-phenylindole) nuclear staining was used for fluorescence

visualization of the cells on the scaffold using fluorescence microscope.

4.2.6 Three dimensional cell culture on polymeric scaffolds in collagen gel and co­

culture with macro phages. B 16 melanoma cells and MCF -7 breast cancer cells were

grown into three dimensional structures on the PDLLA-CTAB scaffold as described above.

Three dimensional growths were maintained in the culture plates till they acquire good

thickness which can be easily visualized under the microscope, usually by day 7-10. Then

collagen gel was made in 24 well plates using 3D collagen cell culture system (Millipore)

using the described protocol. The scaffolds having the three dimensional growth of cancer

cells were gently transferred onto the top of collagen gel and media was added. Growth of

cells was observed over days for evidence of tumor cell migration on the collagen gel. In

some cases the three dimensional growths were embedded in collagen gel and observed for

the tumor cell migration. Cell death in the inner core of the scaffold was visualized by

Propidium Iodide (PI) staining and observation under fluorescence microscopy. Better

tracking of the migrating cells was visualized using Carboxy fluorescein succinimidyl ester

(CFSE) staining. CFSE dye is stably retained within cells and can be used to track them

over days. Co-culture of macrophages and three dimensional cell cultures were also carried

out in collagen gel. THP-1 monocytic cell line was used for the co-culture with cancer cells

and they were initially plated on 24 well plates and cultured. Collagen gel was then made

on top of the THP 1 cells and the cultures of cancer cells are kept on top of the collagen

layer. The THP 1 cells are differentiated into THP 1 macrophages using Phorbol-12-

myrisate-13-acetate (1 0 ng/ml). In wells where the macrophages are to be activated, the

cells were activated using Lipopolysaccharide (1 jlg/ml).

74

4.3 Results

4.3.1 Surface characterization ofPDLLA-CTAB scaffold

Scaffold shape and design guides the proliferating cells to attain proper three dimensional

growth. The scaffold formulated for cell culture was composed of fused PDLLA-CTAB

particles (Fig 4.1 A). Close study of the surface of the scaffold composed of fused

particles using SEM and AFM showed that they are highly porous and rough at nanoscale

(Fig 4.1 B, C). As shown latter, it was seen that after the initial incubation with the cells,

the cells preferentially attaches to crevices of the fused particles, spreads and then

proliferates on the scaffold. The porosity of the particles helps in the free diffusion of the

media in and out of the scaffold and promotes cell growth. The rough topology is suitable

for the cells to anchor, spread and grow on the scaffolds. It has been shown that topology

and patterns at the nano and micro scale influence the behavior of cells in relation to cell

attachment and growth (13, 14). The PLA scaffold thus has the requisite surface

characteristic to promote cell attachment and growth.

4.3.2 Effect of surfactant on cell viability

Surfactants and emulsifiers are widely used in many emulsion based polymer scaffold

fabrication techniques and have significant effect on cell growth. Polyvinyl alcohol

(PVA) is commonly used in many formulations of polymer delivery systems given their

relative biocompatibility. CTAB has also been used in a few drug delivery polymeric

formulations (15). In the present scaffold fabrication process, CT AB, a cationic surfactant

was used to formulate particles which were later fused to form membrane type structure.

It was thus essential to evaluate the cytotoxic effect of CTAB and its residual

concentration on the scaffold. PDLLA polymeric films without any surfactants were

made along with PDLLA films with PV A or CT AB at different concentrations (Fig 4.2A­

C). PV A being hydrophilic remained dispersed as islands in the polymer film while in

case of CTAB, the surfactant dissolved in the organic phase containing the polymer (Fig

4.2B, C). MCF-7 cells were seeded in uniform numbers in all the polymer films and

75

Fig 4.1. SEM and AFM of fused particles (A) SEM image of fused porous particles forming the scaffold. (B) SEM of the particle surface shows that they are highly porous.

Fig 4.1 (C) AFM image of the particle shows that the surface has a rough topology at the nanoscale

76

cultured for a day. Cells were then dissociated from the films and checked for viability

using trypan blue staining. The results of the experiment are shown in Table 4.1.

Presence of surfactant in the polymer film was found to be detrimental to cell growth.

Plain PLA film supported maximum cell growth in comparison to the control well

without any polymer solution. CTAB was highly toxic to the cells in comparison to PLA­

PV A film which supported better cell growth. As seen in Fig 4.2 B, PV A lies as islands

in the PLA film and the cells grow better in the comparatively PYA free regions.

CTAB is used in the preparation ofPDLLA-CTAB particles which are used to make the

scaffolds and it is of concern if the CT AB can be harmful to the cells. When PDLLA­

CTAB particles are wetted with ethanol three main events takes place, (1) Rapid fusion

of PDLLA-CTAB particles into a scaffold (2) Desorption of CT AB molecules from the

surface of particles in presence of ethanol (3) Sterilization of the scaffold in presence of

ethanol. After a few washes with ethanol, it was observed that there was no significant

presence of CTAB molecules in the ethanol. This indicated that most of the CTAB

molecules were removed during washing with alcohol. It was also observed that the

PDLLA-CTAB scaffold after alcohol treatment is suited for the three dimensional growth

of animal cells, indicating the relatively surfactant free PDLLA scaffold.

Control well 105 cells

PLA film 8.3x104 cells

CTAB (0.1%) No viable cells

CTAB (0.25%) No viable cells

CTAB (0.5%) Extensive cell lysis

PVA (0.1 %) 6x 104 cells

PVA(0.25%) 5.2x 104 cells

PVA (0.5%) 3.7x 104 cells

Table 4.1 Effect of surfactant on MCF -7 viability

77

A

50um

Fig 4.2. Microscopic picture of polymer film with surfactant (A) Plain PDLLA polymer film, (B) PDLLA film containing PYA. PYA can be seen lying as island in the polymer film , (C) PDLLA film containing CT AB . CTAB causes formation of vacuoles in the polymer film .

78

4.3.3 Pre-treatment of PDLLA-CT AB scaffold for growth of animal cells

Pre-treatment of PDLLA-CTAB scaffold was optimized for three dimensional growth of

animal cells. Formulated PDLLA-CTAB particles were stored in minus 20°C till needed.

PDLLA-CT AB particles were taken, weighed and spread uniformly in a sterile Petri plate

and wetted with ethanol. In presence of ethanol, the PDLLA-CTAB particles

immediately fused and self-assembled into a polymeric scaffold. The scaffold was

repeatedly washed with ethanol to remove maximum amount of CT AB molecules. The

scaffold was then treated with ethanol for three hours to sterilize it and to remove any

residual surfactant molecules. Afterwards, the alcohol was removed and the scaffold

properly washed with sterile PBS thrice. The scaffold was then broken down into smaller

pieces if needed to make it suitable for cell culture in culture plates. Scaffolds were then

transferred to six well plates containing 15 % of Fetal calf serum (FCS) in RPMI media

and kept in the incubator for overnight treatment. Serum treatment of scaffolds makes the

polymer surface more suitable for cellular attachment by deposition of various serum

proteins like fibronectin and vitronectin (16).

The next step after overnight treatment in serum was the incubation of scaffolds with

cells. Since the scaffolds are kept in culture plates which have surface treated for .cellular

attachment and growth, during incubation most of the cells gets settled and attach to the

culture plates. It was necessary to incubate the scaffolds with a high concentration of

cells for sufficient amount of time for the cells to preferentially attach to the scaffolds.

Too high a concentration of cells for long time can result in cell death. After

experimenting with different concentration of cells for varied times, the micro mass

culture technique by Daniels et al. was modified for initial seeding of our scaffolds (17).

Here, 0.1 million cells in 20111 was incubated with the scaffold for 50 minutes.

Afterwards, sufficient media was added to culture plates and cell culture continued over

days. It is observed that after the incubation with cells, few cells preferentially attach to

the crevices of the scaffold formed by the fused particles. Then over days, the cells start

proliferating over the scaffolds and grew into three dimensional structures. Most of the

remaining cells attach to the bottom of the plate. After two days of culture, the scaffolds

79

are gently transferred to new wells, so that there are no cells growing on the culture plates

and cells grow only in the scaffold. Media was changed on alternate days and culture

continued till required .

4.3.4 Growth of animal cells on polymeric scaffolds

In the field of Biotechnology, animal cell cultures like Chinese Hamster ovary (CHO)

and Vero cells are cultured to produce useful biologicals like monoclonal antibodies,

therapeutic proteins and viral vaccines. Growth of CHO and Vero cells on PLA scaffold

were evaluated. CHO cells attached and grew well on the scaffolds (Fig 4.3 A, B). Vero

cells attached and proliferated on the scaffolds to form three dimensional structures on

the PDLLA-CTAB scaffolds (Fig 4.4 A-D). Three dimensional growth ofthe various cell

lines were observed over days with the help of optical microscope, since it was easy to

use and monitor the growth of cells on the scaffold on a daily basis. But the growth of the

cells could only be observed on the sides of the scaffo ld as the cells growing on top or

bottom are opaque to observation by optical microscope due to the opacity of the polymer

particles. To check whether the attachment and growth of the cells were uniform on the

scaffolds, DAPI nuclear staining and fluorescence microscopy was used for B 16

melanoma cells. These cells were chosen because they were observed to proliferate

rapidly on the scaffolds. Fluorescence microscopy of the cells showed that they were

uniformly spread on the scaffold composed of the fused PDLLA-CTAB particles (Fig 4.5

A-F).

Scanning electron microscopy (SEM) images of attachment and growth of NIH fibroblast

cells on the PDLLA-CT AB scaffolds were also taken to better visualize the interaction

between the cells and scaffold (Fig 4.6 A-D). Fibroblasts are versatile cells and are

known for their capability to have dynamic interactions with the extra cellular matrix and

are involved in the process of wound repair and healing. The SEM images showed that

the fibroblast cells attached to the surface of the scaffolds, spread and proliferate on the

scaffolds, maintaining their characteristic morphology.

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Fig 4.3 A & B. Growth ofCHO cells on the PDLLA-CTAB scaffold

Fig 4.4 Growth ofVero cells on PDLLA scaffold. (A) Vero cells could be seen spreading as a monolayer on the scaffold composed of the fused particles (B&C) By day 4 the cells starts growing into multilayered growth on the scaffold (D) By day 6, thick three dimensional growth of Vero cells could be seen covering the particles.

81

c

Fig 4.5 DAPI staining of B 16 melanoma cells growing on the scaffold. Figures A, C & E shows optical microscopic images of melanoma cells growing on the scaffolds. Cells growing on the boundaries of the particles could be clearly visualized, but other regions are opaque. Figures B, D &F show the corresponding florescent images after DAPI staining. The images confirm the proliferation of B 16 cells at most parts of the scaffold which are not visible in ordinary microscopy.

82

Fig 4.6 Attachment and growth of NIH fibroblast cells on PDLLA-CT AB scaffold. (A) Plain optical microscopy of NIH fibroblast cells on PDLLA -CTAB scaffold. (B) SEM image shows attachment and spread of fibroblast cells on the fused PDLLA particles composing the scaffold. Cells could be seen stretching between the particles.

83

Fig 4.6. Scanning electron micrographs of fibroblasts proliferation on the PDLLA-CT AB scaffolds. (C, D) Cells could be seen attached to polymer surface

84

4.3.5 Three dimensional growth of cancer cells on polymeric scaffolds

Melanoma cancer cell line (B 16) and breast cancer cell line (MCF -7) were grown on the

scaffolds into three dimensional structures. B 16 melanoma cancer cell lines were seeded

on scaffolds as described earlier and the growth of the cells were observed over days. It

was observed that after initial incubation with the scaffolds, few of the cells attached to

different points on the surface of the particles and some preferentially attach to the

crevices formed between the particles (Fig 4.7). The cells after initial attachment on the

scaffold start proliferating and spreading on the particles (Fig 4.8 A). By day 4, the cells

have covered up most of the surface and start growing into multilayered structures as

evidenced by the increase in thickness of the cellular growth (Fig 4.8 B-D). By day 7-8,

three dimensional growth of cells of more than 5jlm and cells have high viability were

observed (Fig 4.8 E). After this time point, it was observed that as the growth becomes

thicker, the inner cell layers become unhealthy and an outer proliferating cell layer can be

clearly discerned (Fig 4.8 F). There was also a rapid increase of necrotic cells. As cells

grow in thickness, diffusion constraints decreased the availability of nutrients to the inner

cell layers and in the body beyond a certain size. Since there is no neo vasculogenesis in

the three dimensional culture, beyond a critical size the inner cell layers undergo necrosis

due to hypoxia and nutrient deficiency.

MCF -7 breast cancer cells were successfully grown on the scaffolds into three

dimensional structures (Fig 4.9 A-D). Thicker growth of cells was observed with MCF -7

cells on the scaffold. The MCF-7 cells were disassociated from the scaffolds at different

time points using Sigma cell dissociation buffer and the viable cell counts were taken

using Trypan blue staining. It was observed that there was steady increase in cell

numbers on the scaffolds over days and the cultures were maintained for about two

weeks. At day 10, the viable cell count per mg of the scaffold was about 0.3 million

MCF- 7 cells (Fig 4.1 0). It was observed that after day 10, there was a steady increase in

the amount of dead cells in the total population of live and dead cells. It is proposed that,

before the critical phase where the inner cell layers start dying, the three dimensional

growth of cancer cells can be used as a model for drug testing, as studies have shown that

they are better model than conventional two dimensional cell culture. The phases of cell

85

growth where the inner cell layers progressively undergoes hypoxic stress and necrosis

can be advantageously used to study certain aspects of tumor biology which are peculiar

to solid tumors like breast cancer is described later.

.....

Fig 4.7 B 16 Cells after incubation with the PDLLA-CTAB scaffold preferentially attach to the crevices formed by the fused particles. The arrow indicates such a cell.

86

Day2 Day4

Day6

DayS

Day 10

Fig 4.8 Growth of B 16 melanoma cells on PLA scaffold. (A&B) By day 2 the B 16 cells have proliferated and covered the scaffold composed of the fused PLA particles. By day 4 the cells starts growing into multilayered structures on the scaffold. (C&D) The images shows the three dimensional growth of cells by day 6 (E&F) By day 8-10, thick three dimensional growth formed by the cells on the scaffold. The arrow in the last image clearly demarcates the dense inner core and the outer surrounding having healthy proliferating cells.

87

Fig 4.9 Three dimensional growths ofMCF-7 breast cancer cells on the PDLLA-CTAB scaffold. (A-D) The images show three dimensional growths on day 5, 10, 13 and 15 respectively. _,

88

3.5

3.0

0> E 2.5 --"' 0 ..--X c 2.0 c ,_ Q) 1.5 ..0 E :J c

1.0 Q)

u 0.5

0.0 3 4

I E22Z1 cell number

5 6 7

Days

8 9 10 11

Fig 4. 10 Viable cells counts ofMCF 7 cells dissociated from the scaffold at different days

89

4.3.6 Three dimensional cancer cell culture on polymeric scaffolds in collagen gel to

study the process of tumor cell migration.

Solid tumors generally have areas of chronic hypoxia due to irregular vasculogenesis and

these inner areas of chronic hypoxia have been shown to influence the process of tumor

cell migration and invasion (18). As observed earlier, the inner cell layers of the growth

of cancer cells on the scaffold undergo progressive cell death, probably due to hypoxia

after a critical period of growth. This model can be advantageous to study certain aspects

of cancer biology especially the relationship between inner developing hypoxia of the

tumor and acquisition of invasion and metastatic ability by the tumor cells. It has been

shown that the role of hypoxia and ECM in the process of tumor cell migration and

invasion is best studied in a three dimensional context (19). Three dimensional cultures

of cancer cells on scaffolds can be valuable tool to study this process as compared to

conventional monolayer cultures.

Preliminary evaluation of tumor cell migration in three dimensional culture was carried

out with B 16 melanoma cell line and MCF-7 breast cancer cell line. B 16 was chosen

because it is known to be an invasive cell line, while MCF-7 does not show much

invasive properties. It would be good to compare the activity of these two cell lines in

three dimensional cultures with regard to tumor cell migration. It is hypothesized that in

the growing tumor, there are areas of hypoxia and this in a way drives the tendency of the

tumor cell migrate and eventually invade the surrounding tissue and extra cellular matrix.

To mimic this process, initially cancer cells were grown on the scaffolds and when the

thickness of the growth is adequately thick they were transferred on to collagen gel and

its behavior observed over days. With progression of cell growth it was observed that the

inner cell mass undergoes cell death. To check this phenomenon, Propidium iodide (Pl)

staining and fluorescence microscopy of three dimensional growths of B 16 cells on

collagen gel was carried out. This confirmed the presence of necrotic cells in the inner

regions of the three dimensional growths (Fig 4.11 A-D).

90

Fig 4.11 Images of B 16 cells showing necrosis. (A) Optical image of the 3D growth of B 16 cells on collagen gel, (B) PI staining of the 3D growth showing presence of large number 'Of necrotic cells in the inner cell mass.

Fig 4.11 (C) Optical image of the 3D growth of B 16 cells on collagen gel, (D) PI staining of the 3D growth showing presence of large number of necrotic cells in the inner cell mass.

91

Three dimensional cultures of B 16 melanoma cells on the scaffolds were placed on

collagen gel and observed over days. Till about a week of growth on the collagen gel, the

three dimensional structures maintained their boundaries and there was no movement of

cells on the collagen gel (Fig 4.12 A, B). By day 8, in certain regions, cellular movement

on the collagen gel was observed and this particular region was observed over days (Fig

4.12 C,O) At the same time, the inner cell mass was observed to be cloudy and was under

hypoxic stress (Fig 4.12 E,F). By day 11 and 12, this region increased in size and

invasive protrusions could be seen (Fig 4.12 G, H). In some areas of the collagen gel,

tumor cell migration and formation of micro growths from the initial three dimensional

growth on the scaffold could be seen (Fig 4.13 A-F).

In order to increase the resistance to tumor cell migration and invasion, the three

dimensional cultures of B 16 cells grown on the POLLA-CTAB scaffold was embedded

in the collagen gel and observed over days (Fig 4.14 A-F). By day 8, definite movement

of cells from the well defined border of 30 growth into the collagen gel could be

observed. Compared to 30 B16 cells on collagen gel, the 30 MCF-7 growth showed little

tendency to migrate on the collagen gel. The three dimensional growth of MCF -7 cells

continued to maintain their cellular boundaries on collagen even after a week of culture I

on the collagen gel (Fig 4.15 A, B). By day 14-16, some micro growths could be seen on

the collagen gel arising from the three dimensional growths, but were very less in number

and nature of aggressiveness, as compared to B16 cells (Fig 4.15 C-F). The three

dimensional model for tumor cell migration and invasion was able to reflect the invasive

and aggressive nature of B 16 cells in comparison to the non invasive MCF -7 cells. The

main objective of these preliminary evaluations is to show that the three dimensional

cultures of an invasive cell line like B 16 on the PDLLA-CT AB scaffold is a versatile

system to be developed into a model for tumor cell migration, invasion and metastases

which could be used as a tool to understand the processes behind this phenomenon and

also as a model to evaluate potential anti metastases drugs. To better track the migration

of the cells on the collagen gel, preliminary evaluation to track the live cells with CFSE

staining were carried out (Fig 4.16).

92

Fig 4.12. Growth ofB 16 cells on collagen gel (A) B 16 cell growth on day 2 on scaffold kept on collagen gel (B) Cell growth on day 3

Fig 4.12 (C) Cells are seen moving out of the well defined boundary onto the collagen gel on day 8 (D) The same area observed on day I 0.

93

Fig 4.12 (E&F) B 16 cell growth at day 10 is viewed at higher magnification to better visualize ~he migrating cell mass on collagen and the inner regions of the 30 growth is seen to be under hypoxic stress and undergoing necrosis

Fig 4.12 (G&H) The same region ofB16 growth as observed on day 11 and day 12. The migrating cell mass on the collagen have acquired invasive protrusions

.; 94

Day7 Day9

Fig 4.13. 3D micro gruwths ofB 16 cells (A&B) B 16 micro growths arising from the 3D 5rowth on scaffolds on collagen gel

0

Day7 DayS

Fig 4.13 (C&D) Two micro growths ofB16 cells in collagen observed over days

Day 10 Day 12

Fig 4.13 (E&F) Two micro growths of B 16 cells in collagen observed over days

•" <.•, ·'

95

-·--

Day3 Day8

Day6 Day9

Day7 Day 10

Fig 4.14 Collagen embedded three dimensional cultures of B 16 melanoma cells. (A&B) Cells are contained within a well defined cellular boundary in the collagen up to day 6, (C&D) ~6 cells could be seen coming out ofthe boundary into the collagen gel by day 7 ,(E&FjBy day 9-10, large population of cells could be seen migrating into the collagen gel.

96

3D MCF 7 growth on collagen, day 2 Micro growths ofMCF 7 on collagen, Day14

3D MCF 7 growth on collagen, day 6 Micro growth ofMCF 7 on collagen, Day 14

Micro growths ofMCF 7 on collagen, Day 14 Micro growth ofMCF 7 on Collagen, Day 16

Fig 4.15 MCF-7 growth on scaffold in collagen gel. (A, B) MCF-7 growth on scaffold showed little tendency to migrate on collagen gel as compared to B 16 cells. (C, D) Few micro growths of MCF -7 which have risen from the growth on scaffolds could be seen by day 14. (E, F) The micro growths ofMCF-7 cells in collagen gel undergo progressive necrosis.

97

Fig 4.16 A. Migration ofB 16 melanoma cells on collagen from 3D growth

Fig 4.16 B. CFSE staining of B 16 cells to track cell migration

98

4.3.7 Co-culture of cancer and macrophages in 3D system

Macrophages in the tumor microenvironment have been' shown to influence the processes

of tumor cell invasion and metastasis and such cells have been designated as Tumor

associated macrophages (TAM) (20). Cancer cells can activate the tumor associated

macrophages to release a wide variety of growth factors, proteolytic enzymes, cytokines

and inflammatory mediators and many of these factors are key agents in tumor cell

migration and invasion (21). Co-culture studies of multicellular tumor spheroids and

macrophages have been carried out to study their interaction and influence on the process

of tumor cell migration and invasion (22, 23). Cancer cell growth on scaffolds are more

stable and easy to handle and here an attempt is made to co culture them with

macrophages in collagen gel as a possible in vitro model to study these interactions.

THP-1 monocytic cell line was plated and cultured on 24 well plates and then collagen

gel was made on top of this cell layer. Three dimensional growth of B 16 cells on

PDLLA-CTAB scaffold was then transferred on the collagen gel and observed over days

(Fig 4.17 A-C). The THP 1 cells were activated to macrophages using PMA (Phorbol

Myristate acetate). By day 2, tumor cells could be seen migrating from the 3D growth

(Fig 4.17 D, E) and at this stage the THP 1 cells were activated by using LPS (Lipo

polysaccharide) (Fig 4.17 F). By day 3, the cells continue to migrate on the collagen gel

and the macrophages could be seen to have activated morphologies (Fig 4.17 G-1). This

experiment was mainly carried out to see if co culture of 3D growth and macrophages

could be achieved in a collagen gel. This could be used to study the effect of immune

cells on processes like migration and invasion in a three dimensional context.

99

Fig 4.17 Three dimensional growth of B 16 cells on collagen gel (A) B 16 growth in collagen with THP 1 monocytes, Day 1 (B) B 16 growth in collagen with THP 1 monocytes, Day 1 (C) THP 1 cells activated with PMA in the collagen gel, Day 1 (D) B 16 growth in collagen with THP 1 monocytes, Day 2, (E) B 16 growth in collagen with THP 1 rnonocytes, Day 2 (F) THP 1 cells activated with LPS in the collagen gel, Day 2 (G) B 16 growth in collagen with THP 1 monocytes, Day 3, (H) B 16 growth in collagen with THP 1 monocytes, Day 3 (I) THP 1 macrophages showing activated morphology

100

4.4 Discussion

In this chapter successful growth of animal on the PDLLA-CT AB scaffolds has been

described. Surface characterization of the scaffolds with SEM showed that fusion

predominantly occurs at the points of contacts between the particles. This ensures that a

porous membrane or scaffold is formed by the spaces between the fused particles and

thus the porosity of the scaffolds can be modulated by using particles of different sizes.

Closer inspection of the particle surface using SEM and AFM revealed that the scaffold

surface is highly porous. Such porosity allows for the free flow of media in and out of the

particles, which may be beneficial for promoting cell growth. Since CTAB is known to

be toxic to cells there was an initial apprehension that it may prove to be toxic to growing

cells. But repeated, thorough washes with ethanol and a three hour treatment removed

majority of the surfactant molecules form the scaffolds. After the ethanol treatment, the

scaffolds were subjected to overnight treatment with fetal calf serum, the main purpose of

which was to allow the scaffold to absorb proteins like fibronectin and vitronectin, which

is beneficial for cell attachment and growth.

Various cell lines like CHO cells, VERO cells, NIH fibroblasts and cancer cell line like

Bl6 melanoma cells and MCF-7 breast cancer cell lines were grown on the scaffolds. It

was observed that the cells attached and proliferated well into three dimensional

structures on the scaffolds. In this study, th~ scaffolds were primarily evaluated for the

three dimensional growth of cancer cells especially B 16 and MCF7 cells. Since it has

been shown that three dimensional growths of cancer cells serve better as an in vitro

model than monolayer cultures (24), it was our interest to use the three dimensional

growth of cancer cells on the scaffold as a model to be used for testing of anti cancer

drugs, as detailed in the next chapter. Preliminary work was also carried out to develop a

model for studying migration of tumor cells by embedding the scaffolds with cancer cells

on collagen gels. Co-culture studies of macrophages and the three dimensional growths

of cancer cells are in progress to study their interactions. Such studies have been shown

to be very important in understanding the process of metastasis oftumor cells (25).

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4.5 Conclusions

The topology of the PDLLA-CTAB scaffold both at the microscopic and macroscopic scale

proved suitable for growth of wide variety of animal cell lines like CHO, Vero, NIH and

other cancer cell lines like MCF 7 and B 16 melanoma cells .. At the micro scale, it was

observed that the surface of the particles had many pores, which may allow free flow of

media thus supporting cell growth. AFM images of the surface of the particles showed that

the surface had a rough topology at the nano scale, suitable for cell attachment and growth.

The ethanol treated PDLLA-CT AB scaffolds were found to be suited for three dimensional

cultures of animal cells. An elaborate treatment protocol was developed to make the

scaffold amenable for cell growth. Within 2-4 days, the attached cells start spreading and

proliferating resulting in a multi-layered growth on the scaffold. By day 8-10, multi-layered

tissue like structures is formed by the cancer cells on the scaffold. No toxicity was

observed with the polymeric membranes formed from the surfactant coated particles and

the cells showed high viability(> 90%) till 7 days, beyond which there were was a gradual

decrease in viability. This may be most probably due to necrosis of cells in the inner region

of growth due to hypoxia and a dense core with surrounding proliferating healthy cells

could be discerned on later stages of three dimensional growth. The cancer cell growth on

the scaffold was kept in collagen gel and this was to develop an in vitro model to study the

process of tumor cell migration and invasion. Preliminary studies involving the co-culture

of cancer growth on scaffolds and macrophages was carried out to study their interaction

and influence on the process of tumor cell migration. PLA scaffold based 3D culture of

cancer cells have multiple applications in cancer biology like study of cell growth and the

effect of microenvironment, migration and invasion of tumor cells, interaction with other

cell types like macrophages and as an in vitro model for anti cancer drug testing.

102

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