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Department of Pharmaceutical Science Thesis Defense Investigator: Bhawani Aryasomayajula

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Department of Pharmaceutical Science Thesis Defense, Northeastern University

Three-Dimensional Spheroid Culture: A Promising Tool for Cancer Research Presented by: Bhawani Aryasomayajula, M.S. Student Date to be presented: 08/23/12 Time & Location: 140 the Fenway, Room 230

Thesis Committee: Chair Dr. Vladimir P Torchilin ___ Member Dr. Rupa Sawant__________ Member Dr. Rajiv Kumar ______ Monolayer cultures have been the preferred method for testing the activity of anticancer drug candidates in-vitro. However, cell based assays performed in monlayer culture provides data from cells in different state lacking many of the characteristics of the tumor environment such as the necrotic core, hypoxic region and low pH at core. A novel approach to overcome the drawbacks of monolayer cultures is the three-dimensional in-vitro culture also knows as multi-cellular tumor spheroids or Spheroid culture or three-dimensional culture. Micro tumor tissues produced by spheroid culture helps to mimic many of the characteristics of tumor environment as well as helps to produce data more relevant to in vivo tumors. In this report, we optimized various techniques of culturing spheroids and worked towards making spheroid culture quick, accurate, and high-throughput. Two methods of spheroid culture were examined, non-adhesive liquid overlay with and without Reconstituted Basement Membrane (RBM / Matrigel) and hanging drop method. Non-adhesive liquid overlay without RBM was found to be the most effective. These spheroids were consistent with respect to size, shape and cell number. The spheroids generated by this method were utilized to evaluate cancer cell behavior in three dimension. To determine the cytotoxicity of paclitaxel in spheroids the spheroid cultures after treatment with a drug, the activity of phosphatase enzyme, using pNPP as the substrate, was measured. The cytotoxicity of paclitaxel in spheroids was then compared to the cytotoxicity in monolayer cultures of A549 cells. The results showed significant reduction in cytotoxicity of paclitaxel on A549 cells when treated in spheroid culture compared to monolayer cultures. Three-dimensional spheroids of A549 cells did demonstrate inherent drug resistance demonstrated by many in vivo tumors. The reduced cytotoxicity of paclitaxel could be due to inhibited drug penetration resulting from cell-cell interaction and presence of cells in different cell cycle stages. It is, therefore, cell cycle analysis was carried out on A549 cells grown as spheroids and compared with cells in monolayer culture using propidium iodide. Results indicated presence of significantly higher population in G0/G1 phase and reduced population in S phase in spheroids compared to monolayer culture. Based on the results obtained, we believe use of spheroids in cancer research could provide a unique platform to conduct previously unfeasible experiments and provide valuable insights for cancer cell behavior in tissue like structure.


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Resources Available Laboratory space: The present study was performed at the Center for Pharmaceutical Biotechnology And

Nanomedicine located at rooms 213, 214 and 215 at Mugar Life Sceinces Building, Northeastern University,

Boston, MA. The facilities have all the necessary reagents as well as equipment with laminar flow for aseptic

work with cancer cells, an ELISA plate reader to acquire readings for ELISA as well as cytotoxicity assays, a

BD FACS (fluorescence activated cell sorter) for analyzing DNA content, fluorescence microscope for taking

pictures of the spheroids in various stages of growth, an HPLC for measuring loading efficiency, chemical

hoods for organic synthesis, salts for the preparation of buffers and a dedicated area for working with animals

with or without the use of radioisotopes. Additionaly, other laboratory facilities on campus are available in

cases where the Center does not have specific equipment (examples: NMR, electron microscope etc).

Laboratory Safety: The investigator has received Laboratory Safety and Chemical Hygiene training parts I and

II, completed in January 2011.


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Biographical Sketch

Bhawani Aryasomayajula Education: 2005-2009: Bachelor of Science, Manipal College Of Pharmaceutical Sciences, Karnataka, India. Major: Pharmacy 2010- Present : Master of Science at Northeastern University, Boston, MA Major: Pharmaceutical Sciences and Drug Delivery Systems Name Academic Affiliation Role Chair: Dr. Vladimir P.Torchilin Department of Pharmaceutical Advisor, Mentor, provides lab space Sciences, Northeastern University and supplies and chair of committee.

Member: Dr. Rupa Sawant Department of Pharmaceutical Reader/ Evaluator Sciences, Northeastern University Member: Dr. Rajiv Kumar Department of Physics, Reader/ Evaluator Northeastern University


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Table of Contents

Page Number

Title Page and Description 1

Resources Available 2

Biographical Sketch & Committee roles 3

Table of Contents 4

1. Introduction 6

2. Background and Significance 8

2.1 Monolayer culture versus Spheroid culture 8

2.2 A brief history of paclitaxel 9

3. Specific Aims 10

4. Material and Methods 11

4.1 Material 11

4.2 Methods 11

4.2.1 Preparation of spheroids. 11

4.2.1.1 Hanging drop method. 12

4.2.1.2 Non-adhesive liquid overlay method with RBM 12

4.2.1.3 Non-adhesive liquid overlay method without RBM 13

4.2.2 Phosphatase Assay method validation to determine viable cells in spheroids 14

4.2.3 Comparison between various methods of cytotoxicity 15

4.2.4 Spheroid size and cell number study 15

4.2.5 Cytotoxicity of paclitaxel in A549 cells (Monolayer vs Spheroids) 15

4.2.6 Cell cycle analysis 16

5. Results 18

5.1 Preparation of spheroids 18

5.1.1 Hanging drop method 18

5.1.2 Non-adhesive liquid overlay with RBM 20

5.1.3 Non-adhesive liquid overlay without RBM 22

5.2 Phosphatase Assay method validation to determine viable cells in spheroids 25

5.3 Comparison between various methods of cytotoxicity 26

5.4 Cell number study in spheroids 27

5.5 Cytotoxicity of paclitaxel in A549 (2D versus 3D) 30

5.6 Cell cycle analysis 31


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6. Conclusion 33

7. Future potential 34

8. References 35


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1. INTRODUCTION

A multi-cellular spheroid culture is gaining increasing interest as a model of choice for cancer research

largely due to limited relevance from monolayer cultures to in vivo studies. To successfully investigate the

behavior of tumor tissues, it is necessary to maintain or recreate characteristics such as cell-cell interaction,

presence of cell population in diverse cell cycle stages depending on available nutrition, low pH as well as

hypoxic region at core level. Monolayer culture, current model of choice, lacks ability to represent any of the

mentioned characteristics. However, multicellular tumor spheroids (MCTS) grown in three-dimension helps

to mimic many characteristics like cell-cell interaction, presence of cell population with diverse proliferation

stage, low pH as well as hypoxic region at core level [1]. Numerous researches have been carried out to

optimize protocol for formation of three-dimensional micro tumor tissue (Table 1).

Table1. List of various methods currently used for spheroid formation.

METHOD ADVANTAGES DISADVANTAGES REFERENCES

Spinner flasks

Simple to perform Massive production Long-term culture Dynamic control of culture conditions Co-culture of different cell types

Require specialized equipment Variation in size/cell number High shear force

2,3,4

Hanging drop

Inexpensive Well controlled spheroid size Fast spheroid formation Co-culture of different cell types Easy to trace spheroid assembly

Labor intensive Massive production difficult

5,6,7,8,9

3D Scaffolds Provide 3D extracellular support

Require specialized equipment for scaffold fabrication

10,11

Pellet culture

Simple to perform Rapid aggregation of a large number of cells

Shear force Massive production difficult

12,13

External force enhancement

Rapid cell aggregation Require specialized equipment and culture conditions

14,15

Non-adhesive surface (liquid overlay)

Inexpensive Simple to perform Easy to scale up

Bulk production possible, Only forms with certain cell types

16,17,18


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Each technique has its own advantages and disadvantages. Non-adhesive liquid overlay technique was used

with few modifications for the formation of spheroids in bulk with uniformity in terms of number of cells as

well as spheroid diameter. This technique resulted in formation of loose clumps or spherical micro tissue

depending on the cell line used. Spheroids formed using such technique can be used to study cellular

behavior, kinetic profiles, gene expression as well as cytotoxicity. Current research on nano-drug delivery

system focuses on numerous studies including cellular association as well as toxicity profile using monolayer

culture. However, monolayer cultures fail to predict tissue penetration [19], which is a limitation factor for

achievement of successful antitumor effect in vivo. A huge disparity has been noticed in the cytotoxicity

profile of a drug between monolayers and in vivo studies. We hypothesize that micro tumor tissues grown

using spheroid culture will mimic various characteristics demonstrated by in-vivo tumors such as cell-cell

interactions, cell population in diverse cell cycle stages and hypoxic core. Therefore, spheroids will help

evaluate cancer cell behavior (response) more accurately than monolayer upon treatment with paclitaxel,

model chemotherapeutic agent.


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2. BACKGROUND AND SIGNIFICANCE

The concept of spheroid culture in cancer research was introduced in 1970 by Sutherland et al., in which

they described the striking morphological similarity between spheroids and certain carcinomas in-vivo with the

help of histological sections [20]. The studies by Moscona and Moscona [21] and Holtfreter [22] also

demonstrated a similar multi-cellular spheroid model in the 1970s, using a self-assembly process from

suspended cells. Since then, the classical system of Sutherland and co-workers of spherical multi-cellular

cultures of animal cell lines has been extended and many research papers use spheroid culture as their primary

research model.

2.1 Monolayer culture versus Spheroid culture

A spheroid above 500µm diameter commonly exhibits characteristics such as a necrotic core surrounded

by a layer of quiescent cells and an outermost layer of rapidly multiplying cells [23]. This morphology is similar

to avascular early stage tumors. Other similarities between the original tumor and the respective spheroids

include volume growth kinetics, differentiation characteristics such as the development of specific histological

structures or the expression of antigens [24]. Several years ago it was established that the observed resistance to

ϒ-irradiation of V79 cells exposed in spheroids compared to cells exposed as single cells (in suspension or in

monolayer), could not be explained solely by the presence of a resistant hypoxic cell fraction in the spheroids

[25, 26]. It was suggested that the high radio resistance of cells irradiated in spheroids was partly due to

intercellular contact and/or biochemical communication in the spheroid. In 1977, Lucke-Huhle & Dertinger

presented data demonstrating that V79 cells in spheroids were protected against hyperthermic damage when

compared to exponentially growing cells in monolayer culture [26]. A genome wide gene-expression analysis of

porcine hepatocytes revealed that more than 65 genes including those encoding hepatic specific transcription

factors, express differently in spheroids thereby making the spheroids resemble more closely to liver tissue than

monolayer hepatocytes [27,28]. Many more such studies have established successfully that numerous

morphological and biochemical characteristics specific to tumors are mimicked by spheroids but are lost in a


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monolayer culture. Multicellular tumor spheroids (MCTS) contain an extensive extracellular matrix (ECM) and

possess a complex three-dimensional network of cell-to-matrix and cell-to-cell interactions. This not only

affects the distribution and function of biological response modifiers such as hormones and growth factors but is

also relevant to the penetration and action of drugs thereby making spheroids, a better model than monolayers

for cancer studies.

2.2 A brief history of paclitaxel

Paclitaxel (Taxol) is a compound extracted from the Pacific yew tree Taxus brevifolia with

antineoplastic activity. Paclitaxel binds to tubulin and inhibits the disassembly of microtubules, thereby

resulting in the inhibition of cell division. This agent also induces apoptosis by binding to and blocking the

function of the apoptosis inhibitor protein Bcl-2 (B-cell Leukemia 2) . Paclitaxel is approved to be used alone or

with other drugs to treat AIDS-related Kaposi sarcoma, Breast cancer, Non-small cell lung cancer and ovarian

cancer. It is also being studied in the treatment of other types of cancer [29].

Figure 1: Structure of paclitaxel


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3. SPECIFIC AIMS

Major objective for the present study was to evaluate and optimize protocol for the formation of

spheroids using A549 cells and MCF-7 ADR cells taking into consideration, the ease of preparation and

consistency with respect to size, shape and cell density. Spheroids, formed using optimized protocol, were used

for testing the cytotoxicity of model anti-neoplastic drug i.e. paclitaxel. Cytotoxicity was measured using

phosphatase assay. This will be investigated by quantifying the amount of cytoplasmic phosphatases which are

released after cell lysis. These results were compared against the cytotoxicity of paclitaxel in monolayer. To

study the reasoning behind any change in the cellular response upon paclitaxel treatment in monolayer and in

spheroid form, a cell cycle analysis was performed. By using the nucleic acid stain propidium iodide to measure

the total DNA content in a cell, the difference in the ratio of cells in various stages of cell cycle was measured,

between spheroids and monolayers.


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4. MATERIALS AND METHODS

4.1 Materials

MCF-7ADR, a multi-drug resistant human, breast cancer cell line was a kind gift from Dr. Minko from

Rutgers University (NJ, USA). A549, a human lung adenocarcinoma cell line was obtained from ATCC. Both

cell lines were maintained at 37 0C in a 5% CO2 incubator in all experiments in DMEM supplemented with 10%

Fetal Bovine Serum (FBS) and 1 % antibiotic solution (10,000 I.U./ml Penicillin, 10,000 µg/ml Steptomycin,,

25ug/ml Amphotericin B). Protein electrophoresis grade Agarose (high gelling temperature) was obtained from

Fisher Scientific (NJ, USA), p-nitro phenyl phosphate (pNPP) substrate was obtained from Thermoscientific

(IL, USA), Sodium acetate and Triton X-100 were obtained from Sigma Aldrich (MO,USA). Reconstituted

basement membrane (matrigel) (RBM), high concentration was obtained from BD Biosciences (MA, USA).

Propidium iodide was obtained from Invitrogen (CA, USA) and RNAse A was obtained from Quiagen (CA,

USA). Accutase was obtained from Innovative Cell Technologies (CA, USA). Cell-titer blue cell viability assay

kit and LDH release assay kit (Cytotox 96 non-radioactive cytotoxicity assay) were obtained from Promega

(WI, USA).

4.2 Methods

4.2.1. Preparation of spheroids

The fundamental requirement for the formation of spheroid is to prevent the attachment of cells to the

surface on which they are growing. This should facilitate cell-cell interaction with the neighboring cells and

ultimately to form a three-dimensional structure. The formation of the three dimensional structure will be

unique depending on the method used and the cell line used. The aim was to find a method that resulted in

formation of consistent spheroids in terms of shape, size and density. Apart from these requirements, the

method, after optimization had to be high throughput and the format of the method conducive to modifications

for the purpose of various studies. Out of numerous methods in practice, hanging drop method, non-adhesive

liquid overlay method with or without reconstituted basement membrane (RBM) were evaluated for the


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formation of spheroids because these methods could be optimized to be high-throughput. The spheroids

obtained from these methods were checked for consistency in shape, size and number of cells to evaluate and

select the most efficient method. To determine consistency in size and shape, pictures of the spheroids were

taken using Nikon Eclipse E400 microscope at exposure 251.3 and gain 4. To determine consistency in number

of cells, phosphatase assay was performed on various days.

4.2.1.1. Hanging drop method

A549 cells were grown in monolayer till 70% confluent. Cells were then trypsinized and centrifuged at

1400 rpm for 4 minutes to get cell pellet. Cells were re-suspended in complete DMEM followed by cell

counting using trypan blue exclusion method. Cell suspension was adjusted to 10,000 cells / 20 µl in complete

media. Using a multi-channel pipette, 20 µl of the cell suspension was deposited on the lid of a 96-well plate in

the center of each groove that fits on top of a well. The plate was inverted over a 500 cm2 sterile covered cell

culture plate containing 100 ml of media to avoid dehydrating the drops and maintain moisture levels. Cell

aggregation was allowed to occur for 3 days, due to gravity, which initiated spheroid formation. The well

established cell aggregate in each drop was transferred to corresponding wells, pre-coated with 50 µl of 1.5%

agar in serum free DMEM and containing 150µl of complete DMEM. The transfer was achieved by carefully

placing lid of the plate on the plate and centrifuging it at 1200 rpm for 4 minutes at 25°C to achieve a smooth

transfer of spheroids without physical contact. The 96-well plate containing these spheroids was incubated at

37oC for further growth of spheroids. Formation of spheroids using this modified hanging drop method was

evaluated for consistency with respect to size and shape. Pictures of the spheroids were taken for 20 days to

observe the aforementioned parameters.


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4.2.1.2. Non-adhesive liquid overlay method with RBM

One of the more popular approaches to spheroid formation is to prevent cell adhesion to the surface on

which they are growing. This should result in the initiation of cell-cell interactions and spheroid formation.

External stimulation can also be provided by the addition of RBM. Addition of RBM supplements extracellular

matrix, which helps to initiate spheroid formation [30]. This approach towards spheroid formation was analyzed

on A549 cell line.

Each well in 96-well plate was coated with 50µl of sterile 1.5% agar in serum free media (DMEM for

A549) for the purpose of providing a non-adherent coating. Addition of 50 µl of agar is important to cover

complete surface of a well and formation of concave surface. Care was taken to keep the temperature of the

sterile 1.5% agar above 65°C to prevent it from gelling during the transfer. After the coating, the plates were

allowed to cool for 40 minutes before the addition of cell suspension. A549 cells were grown in monolayer till

they were 70% confluent. Cells were then trypsinized and centrifuged at 1400 rpm for 4 minutes to get a cell

pellet. The pellet was re-suspended followed by cell counting using trypan blue exclusion method. Cell

suspension was adjusted to 100,000 cells/ml with 1.5% v/v RBM in complete media. For the formation of a

spheroid in each well, 100µl of this suspension was added to pre-coated wells. Plates were then centrifuged at

1500 rcf for 15 minutes at 4oC. Centrifugation resulted in the formation of a single layer of cell pellet. 96-well

plate containing such single layered cell pellets was incubated at 37oC for the formation of spheroids. Formation

of spheroids using this non-adhesive liquid overlay method was evaluated for consistency with respect to size

and shape.

4.1.1.3 Non-adhesive liquid overlay method without RBM

The method described for non-adhesive liquid overlay method with RBM is kept the same except no

addition of RBM to the media while seeding the cells. Seeding and centrifugation is done at room temperature.

Formation of spheroids was observed for 12 days. A549 cells as well as MCF-7ADR cells were evaluated for

consistency with respect to size, shape and number of cells.


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4.2.2. Phosphatase assay method validation to determine viable cells in spheroids

There are numerous methods to determine viable cells in monolayer out of which Cell titer-Blue cell

viability assay is frequently used. However, the use of this method would be inappropriate to determine viable

cells in spheroid culture as it is not validated by manufacturer in spheroids. Therefore, phosphatase assay [31]

was evaluated to determine viable cells in spheroid culture. The amount of phosphatase, cytoplasmic enzyme, is

directly proposal to the viable cells. Cells present in spheroids are rapidly lysed with 1% Triton X-100 to release

phosphatase enzyme. Phosphatase when incubated with p-nitrophenyl phosphate (pNPP), converts pNPP into p-

nitrophenol. After incubation, addition of 1 N sodium hydroxide creates alkaline conditions to convert p-

nitrophenol to p-nitrophenolate. P-nitrophenolate has strong absorbance at 405 nm wavelength. Absorbance at

405 nm wavelength is directly proportional to p-nitrophenolate which corresponds to the amount of phosphatase

and the amount of phosphatase is directly proportional to viable cells.

To determine the working range of phosphatase assay with respect to the number of cells for A549 and

MCF-7ADR the following procedure was carried out. A549 and MCF-7ADR cells were grown in monolayer till

70% confluent. Cells were then trypsinized and centrifuged at 1400 rpm for 4 minutes to get cell pellet. Cells

were resuspended in PBS pH 7.4 followed by cell counting using trypan blue exclusion method. Cell suspension

was adjusted to 2 X 106 cells/ ml with PBS. Cells were adjusted at different cell density ranging from 100,000

cells/100 µl, 50,000 cells/100 µl, 25,000 cells/100 µl, 12,500 cells/100 µl, 6,250 cells/100 µl, 3,125 cells/100 µl,

1,562 cells/100 µl, 781 cells/100 µl, 390 cells/100 µl, 185 cells/100 µl, and 92 cells/100 µl of PBS (n=8).

Immediately, 100 µl of freshly prepared phosphatase assay buffer (2 mg/ml of pNPP in 0.1 M sodium acetate

pH 4.8 with 1% triton X-100) was added to each well followed by 90 minutes incubation at 37 oC. After

incubation, reaction was stopped with 10 µl of 1 N sodium hydroxide. Absorbance was read within 10 minutes

at 405 nm after addition of sodium hydroxide using Biotek – Synergy HT absorbance plate reader.


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4.2.3 Comparison between various methods of cytotoxicity

In order to use the phosphatase assay for determining the cell viability in spheroids, it was first

compared to two other popular methods of assessing cell viability i.e the Cell titer blue cell viability assay

(Promega) and LDH release assay. To determine cytotoxicity of paclitaxel using all three assays, 10,000 cells /

100 µl / well of MCF-7 ADR cells were seeded 24 hours before treatment. Cells were then treated with various

concentrations of paclitaxel and incubated for 48 hours. After incubation cytotoxicity was determined using all

three assays according to manufacturers protocol.

4.2.4 Spheroid size and cell number study

For evaluating the consistency of the spheroids in terms of size, A549 and MCF-7 ADR spheroids were

seeded using non-adhesive liquid overlay without RBM. Pictures were taken everyday and the diameter of the

spheroids was measured using the software Spot Advanced. For checking the consistency of the spheroids

seeded by non-adhesive liquid overlay without RBM with respect to the number of cells, spheroids were

evaluated for a period of 9 days for the number of cells. Spheroids of A549 cell line were seeded using non-

adhesive liquid overlay in complete DMEM. They were allowed to grow till day three after which every

alternate day, starting from day 3, they were withdrawn from the plate, transferred to a fresh 96 well plate,

washed twice with 100 µl of PBS (pH 7.4) and incubated with 100 µl of PBS and 100 µl of phosphatase assay

buffer (pH 4.8) for 90 min. A standard curve for A549 cells in suspension was also seeded starting from

100,000 cells and serial diluted eight times (to 781 cells) and incubated with PBS (Ph 7.4) and phosphatase

assay buffer (pH 4.8) for 90 min. After incubation 10 µl of the stop solution (0.1 N NaOH) was added and the

absorbance was measured at 405nm using a Biotek Synergy HT absorbance plate reader.


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4.2.5. Cytotoxicity of paclitaxel in A549 cells (Monolayer vs Spheroid culture)

For cells in monolayer form, A549 cells were seeded in 96-well plate at a density of 5000 cells / well, 24

hrs before treatment. Cells were treated at various concentrations of paclitaxel and allowed to grow for 48 hrs.

After incubation, cells were washed with fresh media and cytotoxicity was measured using Cell Titer Blue

assay according to the manufacturer’s protocol. Briefly, washed cells were incubated for 2 hrs with 100 µl of

fresh media containing 20 µl of Cell Titer Blue reagent. After the incubation, plates were analyzed for the

fluorescent signal at 530 nm excitation and 590 nm emission wavelengths. Cells treated with vehicle (PBS)

were taken as controls to calculate % cell death and the assay was carried out in triplicate. The curve that was

obtained was analyzed and four concentrations of the drug corresponding to the beginning of exponential part

(3.12nM), IC50 value for monolayers (29.8nM), beginning of saturation (100nM) and a very high concentration

(2500nM) were selected and the spheroids were treated with these four concentrations in the following manner.

For cells in spheroid form, formation of A549 micro tumor tissues were initiated using non-adhesive

liquid overlay method without RBM 72 hrs before treatment. Spheroids were treated at the aforementioned

concentrations of paclitaxel and allowed to grow for 48 hrs. After incubation, spheroids were transferred in

uncoated 96-well plate and washed with PBS. After washing, 100 µl of PBS was added to each well followed

by addition of 100 µl of phosphatase assay buffer (2 mg/ml of pNPP in 0.1 M sodium acetate pH 4.8 with 1%

triton X-100). Spheroids were incubated for 90 minutes followed by addition of 10 µl of 1 N sodium hydroxide.

Absorbance was measured within 10 minutes at 405 nm wavelength after addition of sodium hydroxide using a

Biotek Synergy HT absorbance plate reader.


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4.2.6 Cell cycle analysis:

Spheroids of the cell line A549 cells were cultured using non-adhesive liquid overlay method in 96-well

plates. They were grown till day 6 after which they were withdrawn from the plates into a 15ml tube containing

5 ml of accutase (a cell detachment solution milder than trypsin). The spheroids were allowed to disperse into a

single cell suspension for about 20 minutes at 37 OC. The resulting cell suspension was centrifuged and the

accutase was removed by washing twice with PBS. The cells were counted and adjusted to 1 million cells per

ml of PBS. 100% Ethanol previously cooled by storage at -20 OC was added to the cell suspension slowly with

constant mixing to expose all cells to fixing solution (3ml ethanol per ml of cell suspension). A549 cells grown

in monolayer at 60% confluency were detached from the flask using Accutase and the cells were centrifuged,

re-suspended to 1 million cells per ml PBS and fixed similarly with 100% Ethanol. Both solutions were left

overnight in 4 OC for fixation. The fixed cells were washed with PBS twice to remove all traces of ethanol. Both

tubes were treated with 1ml of 40 µg per ml propidium iodide solution containing 5 µl of 10ug/ml RNAse A per

million cells . Both tubes were incubated for 40 minutes at room temperature. After 40 minutes, both treatments

were analyzed using BD FACScalibur for PI signal.


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5. RESULTS

5.1 Preparation of spheroids

5.1.1 Hanging drop method

A549 cells were evaluated for the formation of spheroids using hanging drop method. After the

incubation of 10,000 cells in 20 µl drop for 3 days, spheroid formation was initiated. Transfer of this spheroid is

required as 20 µl of complete media does not provide enough nutrition for the cells. Transfer procedure was

modified to minimize disruption of cell aggregates by physical shear, labor requirement and increase production

capacity. Intact spheroids were transferred using centrifugal force. After transfer, spheroids continued to grow

until day 15 after which cells started to shed from outer layer. However, formation of spheroids with A549 cells

using hanging drop method resulted in inconsistent spheroids with respect to their shape as can be seen in Fig 2.

2 which makes the use of this method inappropriate. It was, therefore, necessary to evaluate other methods for

the formation of spheroids with consistency.


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Figure 2: Spheroid formation using hanging drop method. 20 µl of cell suspension containing 10,000 A549 cells were hung for 3 days on the cover of a 96-well plate after which they were transferred to wells coated with 50 µl 1.5% w/v agar and 150 µl complete media. The growth of spheroids was observed for several wells for 20 days to determine consistency.


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5.1.2 Non-adhesive liquid overlay method with RBM

A549 cells were evaluated for the formation of spheroids using liquid overlay method with RBM. After

the incubation of 10,000 cells / well, cells initiated formation of spheroids. However formation of spheroids was

from outside to inside part of cell aggregation as can be seen in figure 4. Consistency with respect to size and

shape was achieved, however density of the spheroid remained inconsistent. Intact spheroid forms around day 2

and continues to grow till day 10. Due to varied density from spheroid to spheroid this method would not be

utilized to form spheroids for further experiments. However this method is very useful to form spheroids for the

cell lines that fail to produce spheroids using non-adhesive liquid overlay method without RBM [30].


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Figure 3: Spheroid formation using non-adhesive liquid overlay with RBM. 100 µl of cell suspension in complete media supplemented with 1.5% w/w RBM containing 10,000 A549 cells were centrifuged at 1500 rcf for 15 minutes in a 96-well plate coated with 50 µl agar / well at 4 OC. The growth was observed for several wells for 20 days.


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5.1.3 Non-adhesive liquid overlay method without RBM

A549 cells were evaluated for the formation of spheroids using non-adhesive liquid overlay method

without RBM. After the incubation of 10,000 cells/well, the cells initiated formation of spheroids. A549 cells

when seeded using non-adhesive liquid overlay method without RBM resulted in formation of consistent

spheroids with respect to their size, shape and density as shown in figure 4. Intact spheroid forms around day 3

and continues to grow till day 10. However cells start to shed after day 7. Treatment for further experiments will

be carried out during exponential growth phase, which was determined to be from day 4 to day 7.

To evaluate the broad applicability of this method, we also evaluated the spheroid formation by MCF-

7ADR cells. As can be seen in Figure 5, consistent spheroids were formed with respect to size, shape and

density. However the spheroids can be used for further treatments till day 7 and the cells start to shed around

day 9.


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Figure 4: Spheroid formation using non-adhesive liquid overlay method without RBM. 100 µl of cell suspension in complete media containing 10,000 A549 cells were centrifuged at 1500 rcf for 15 minutes in a 96-well plate coated with 50 µl 1.5% w/v agar per well. The growth was observed for several wells for 12 days to determine consistency. (* __ = 100 µm )


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Figure 5: Spheroid formation using non-adhesive liquid overlay method. 100 µl of cell suspension in complete media containing 10,000 MCF-7ADR cells were centrifuged at 1500 rcf for 15 minutes in a 96-well plate coated with 50 µl 1.5% w/v agar per well. The growth was observed for several wells for 9 days to determine consistency. ( __ = 100 µm)


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5.2 Method validation to determine viable cells in spheroids

A549 and MCF-7ADR cells were evaluated for the correlation of the amount of phosphatase with

number of cells. Phosphatase activity was determined using phosphatase specific substrate pNPP. Using the

conditions mentioned in procedure, number of viable cells could be determined accurately up to 100,000

cells/well for both cell lines.

Figure 6: Phosphatase assay method validation to determine viable cells in spheroids for A549 cells. Several concentrations of cells were seeded in 96-well plate and treated with phosphatase assay buffer on the same day. The reaction was stopped using 0.1 N sodium hydroxide and the absorbance from each well containing different number of cells was measured at 405 nm. (n=8)

Figure 7: Phosphatase assay method validation to determine viable cells in spheroids for MCF-7 ADR cells. Several concentrations of cells were seeded in 96-well plate and treated with phosphatase assay buffer on the same day. The reaction was stopped using 0.1 N sodium hydroxide and the absorbance from each well containing different number of cells was measured at 405 nm. (n=8)

y = 4E-‐05x -‐ 0.0027 R² = 0.99617

-‐0.5 0

0.5 1

1.5 2

2.5 3

3.5 4

4.5

0 20,000 40,000 60,000 80,000 100,000 120,000

Absorban

ce

No. of cells

Validation of cytotoxicity method for A549

y = 1E-05x + 0.1135 R² = 0.99979

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 20000 40000 60000 80000 100000 120000

Abs

orba

nce

No. of cells

Validation of cytotoxicity method for MCF-7 ADR


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5.3 Comparison between the different assays of cytotoxicity

The cell titer blue assay, phosphatase assay and LDH release assay were evaluated for determination of

cytotoxicity of paclitaxel on MCF-7ADR cells in monolayer culture. The comparison between these three assay

methods of cytotoxicity revealed that the cytotoxicity in monolayer could be determined accurately using any of

these methods as shown in fig 8. However, phosphatase assay method was used for cytotoxicity determination

in spheroid culture as it uses cell lysate to determine presence of live cells than metabolically intact cells.

Figure 8: Comparison of cytotoxicity of paclitaxel on MCF-7 ADR cell line determined using cell titer blue, phosphatase assay and LDH release method. 5000 cells / 100 µl / well of MCF-7 ADR cells were seeded 24 hrs before treatment. They were treated with various concentrations of paclitaxel and cytotoxicity was measured after 48 hrs.

-2

0

2

4

6

8

10

-20

0

20

40

60

80

100

-1 -0.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Fold

incr

ease

in L

DH

rel

ease

% c

ell d

eath

Log Conc of Paclitaxel in nM

Cytotoxicity of Paclitaxel in MCF-7ADR at 48 hrs

Cell titer blue assay Phosphatase assay


27

5.4 Spheroid size and cell number studies

The spheroids obtained by non-adhesive liquid overlay without RBM method were evaluated for cell

number and size over a period of time. The size for the spheroids of cell lines A549 and MCF-7 ADR were

measured using Spot advanced software. As non-adhesive liquid overlay without RBM produced consistent

spheroids with respect to size and shape, the number of cells were determined only for A549 cell spheroids

grown using non-adhesive liquid overlay method without RBM method. Determination of the number of cells

was carried out on every alternate day for a period of 9 days using the phosphatase assay method.

The pictures showed that the spheroids are initially bigger in diameter. They continue to shrink over

time and become denser. The diameter decreases continuously for the first few days and then stabilizes. As can

be seen in figure 9, the spheroids grown using non-adhesive liquid overlay without RBM were consistent in

terms of diameter for A549 and MCF-7 ADR cell lines. Also, cell number study showed that the growth of

spheroids, determined using number of cells in the spheroids formed using non-adhesive liquid overlay without

RBM, were consistent demonstrating uniformity among spheroids making them feasible for in vitro analysis

(Figure 10). It was also found that the growth of A549 cells in spheroid was much slower than cells grown in

monolayer culture.


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Fig: 9: Diameter of spheroids from cell lines A549 and MCF-7 ADR over a period of time. Pictures of the spheroids (n = 3) were taken everyday and the spheroid diameter was measured using Spot advanced software.

0

200

400

600

800

1000

1200

1 2 3 4 5 6

Spheroid Diameter (in

Time ( in days)

A549 Spheroid Size Analysis

0 100 200 300 400 500 600 700 800

1 2 3 4 5 6

Spheroid Diameter (in

Time ( in days)

MCF-7 ADR Spheroid SizAnalysis


29

Figure 10: Cell number study for A549 spheroids. Spheroids of A549 cell line were seeded using non-adhesive liquid overlay without RBM. After the initiation of spheroid formation (day3) spheroids (n=3) were withdrawn every alternate day and analyzed for cell number using phosphatase assay method for 9 days.

0

5000

10000

15000

20000

25000

Day 3 Day 5 Day 7 Day 9

cell

num

ber

Time (in days)

Cell number study for A549 spheroids


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5.5 Cytotoxicity of paclitaxel in A549 (2D versus 3D)

The A549 cells grown in monolayer culture were treated with various concentration of paclitaxel and

cytotoxicity was determined using cell titer blue. Dose response curve obtained was used to select various

concentration of paclitaxel for treatment on A549 cells grown in spheroid culture. The comparison revealed that

the cytotoxicity of paclitaxel was significantly lesser when treated with 100 nM and 2500 nM in spheroid

culture than in monolayer culture. Also, Addition of paclitaxel more than 100 nM failed to achieve higher

cytotoxicity demonstrating inherent resistance by tissue structure. Observed significant inhibition in cytotoxicity

and saturation at 50% cell death could be due to limited penetration of paclitaxel and/or presence of cells in

diverse cell cycle stages than monolayer culture.

Figure 11: Cytotoxicity of paclitaxel in A549 cells in monolayer culture. Cell-suspension containing 10,000 A549 cells per 100 µl was seeded in a 96 well plate and allowed to grow for 24 hours. Cells were then treated with various concentrations of paclitaxel for 48 hours. Cytotoxicity was evaluated using Cell-titer Blue Cell viability assay.

-‐10

0

10

20

30

40

50

60

70

80

-‐1.5 -‐1 -‐0.5 0 0.5 1 1.5 2 2.5

Cel

l dea

th (%

)

Log concentration of Paclitaxel (in nM)

% Cell Death In A549 Monolayer


31

Figure 12: Comparison of cytotoxicity of paclitaxel between monolayers and spheroids of A549 cells. Cell suspension containing 10,000 cells / 100 µl of complete media were seeded in the form of monolayers and spheroids (using non-adhesive liquid overlay without RBM) in 96-well plates. Monolayers on day 2 and spheroids (n = 5) on day 4 were treated with four different concentrations of paclitaxel and its cytotoxicity was compared in terms of cell death (%) ( * p < 0.05)

0 10 20 30 40 50 60 70 80

3.1258 29.86 100 2500

% Cell Death

Concentration of Paclitaxel (nM)

Comparison of Cytotoxicity of paclitaxel between A549 monolyers and spheroids at 48 hrs

MONOLAYER

3D


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5.6 Cell cycle analysis

Most of the chemotherapeutic agents including paclitaxel act on rapidly dividing cells. Therefore, cell

cycle analysis was performed on cells grown in spheroids and compared with cells in monolayer culture. To

compare cell cycle stages, cells in growth phase in monolayer culture and spheroid culture were used. Cell cycle

analysis of the monolayers (at 60% confluency) and spheroids of the A549 cells (day 6) revealed presence of

cells in diverse cell cycle stages. In spheroids, cells found in G0/G1 phase were significantly higher than

monolayer culture demonstrating higher cell population in resting phase. Spheroid culture also demonstrated

significantly lower cells in S phase demonstrating lower cell population in synthesis phase. No significant

different was observed in M phase cells (Figure 13). Higher population in resting stage and lower population in

rapidly dividing cells could be one of the many possible reason for reduced cytotoxicity of paclitaxel on A549

cells grown in spheroid culture than in monolayer culture.

Figure 13: Cell cycle analysis of A549 cells in monolayers and spheroids. A549 cells were grown in monolayers (60% confluency) and spheroids (6days) after which they were harvested and fixed with absolute alcohol. They were stained with propidium iodide and fluorescence (FL1-A) was analyzed using FACS. The histogram obtained was analyzed for obtaining the percentage of cells in each phase of the cell-cycle and compared for monolayers and spheroids. ( * p < 0.05


33

6. Conclusion

The use of three-dimensional multi-cellular tumor tissues or spheroids have gained immense popularity

in recent times due to a better replication of the tumor model as compared to monolayer cultures. In this study,

various popular methods of spheroid formation were evaluated to select the most efficient method for

formation of consistent spheroids with respect to size, shape and number of cells. Hanging drop, Non-adhesive

liquid overlay without and with RBM were all evaluated and Non-adhesive liquid overlay without RBM was

found to be efficient, quick and high-throughput. It resulted in generation of spheroids with a uniform shape,

size and density. It involved a 96-well format, making it simple and easy to modify and treat with model drugs

for further studies. The spheroids obtained by this method were subjected to treatment with the free drug

paclitaxel to compare the difference in cytotoxicity between monolayers and spheroids at a given concentration

of drug. For this purpose, the cytotoxicity protocol was changed from the cell-titer blue protocol generally used

on monolayers to one which involved the use of cell lysate of the spheroids to reach cells present at the core of

the spheroid and access the enzymes present in the cytosol. The protocol involving pNPP as a substrate was

optimized and adapted for the purpose of this study by establishing linearity between the absorbance and the

number of cells in MCF-7ADR and A549 cells. After optimization, this protocol was used to evaluate the

difference between the cytotoxicity in monolayers and spheroids upon treatment with similar doses of the drug.

It was found that the cytotoxicity of a drug on spheroids was significantly lesser than its cytotoxicity in

monolayers. To understand the reason behind this disparity, a cell cycle analysis was conducted for the cells in

monolayer culture versus spheroid culture. A difference in the ratios of G°/G1 : S : G2/M was noticed with the

proportion of cells in the G0/G1 in spheroids being significantly higher and the S phase, significantly lower.

Many anti-neoplastic drugs primarily act on rapidly dividing cells. The low proportion of cells in their S phase

in a spheroid could be one of the many possible reason for the observed reduced toxicity. Based on these

results, we can state that spheroid culture is a consistent, high throughput technique that, due to its closer

resemblance to an in-vivo tumor, is a better model for cancer research over monolayer cultures.


34

7.Future potential

Spheroid culture has been shown to be vital in the field of cancer research. Apart from cytotoxicity and

cell cycle studies, it can be used for many other purposes such as to study the role of cell-cell interactions and

cell-matrix interactions, secondary screening of drug molecules, drug and nano-particle penetration, effects of

active targeting in tissue penetration etc. They can be used for determining genetic changes and change in

protein expression profile. Tumor xenografts which are difficult to establish in-vivo can be studied using

spheroid culture. Spheroid culture has the potential for application in fields other than cancer research as well

and therefore is versatile and has immense future potential.


35

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