WO2013050921A1

WO2013050921A1 - Hollow polymer microspheres as three-dimensional cell culture matrix

Info

Publication number: WO2013050921A1
Application number: PCT/IB2012/055251
Authority: WO
Inventors: Jayant Jagannath Khandare; Archana Jalota-Badhwar
Original assignee: Piramal Enterprises Limited
Priority date: 2011-10-03
Filing date: 2012-10-01
Publication date: 2013-04-11
Also published as: TW201335368A; AR088106A1

Abstract

The present invention discloses a hollow polymer microsphere made of biodegradable polymer that is used to generate three-dimensional cell scaffolds. The present invention also relates to process(es) for the preparation of an engineered cell culture matrix comprising hollow polymer microspheres. A process of screening anticancer therapeutic agents by growing myeloid cells in an engineered cell culture matrix comprising hollow polymer microspheres is also provided.

Description

Hollow Polymer Microspheres as Three-Dimensional Cell Culture Matrix Field of the invention The present invention provides an engineered cell culture matrix suitable for growing cells in hollow polymer microspheres which have the ability to cause monolayers to form three-dimensional scaffolds. The three-dimensional scaffolds formed can be used in screening. Background of the invention

Proliferation and differentiation of cells in culture milieu holds the key for basic cancer research and is finding more applications in drug discovery research. Several factors contribute to proliferation and differentiation of cells in vivo, especially in a diseased condition and replication of identical physiological conditions in vitro to test potential therapeutics has been a challenge. The physical aspects of the cells in culture such as composition, architecture and cell-cell interaction are extremely important while recreating cells' natural environs. Towards this direction, continuous attempts are being made to develop matrix with three-dimensional (3D) architecture as against conventional two dimensional (2D) culture systems to better mimic the physiological changes that occur in vivo during diseased conditions.

Cell culturing technologies have gradually progressed from catering to academic interest to satisfying industry and commercialization needs, especially to provide culture systems to test drug efficacy and toxicology in vitro. To this end, various 3D cell culture technologies have been explored and put to use. Conventionally, the most useful technology has been the hanging drop wherein embryonic, tumor and primary cells have been grown in vitro. However, one drawback of this technology has been the inability to produce them for high throughput needs. In addition, the hanging drop system achieves a maximum cell size of 200μΜ which may be an impediment for studying the mechanical activity of a cell in response to a test therapeutic. Furthermore, InSphero, a supplier of biological microtissues, has come up with three-dimensional tumor microtissues that enable mass production to test the drug efficacy in 3D disease models.

US Patent Application US20080194010, provides 3D inserts made of nonbiodegradable and non-cytotoxic polymers with an internal and external space for living cells to attach, proliferate and differentiate. These inserts however need to be used with tissue culture plates or flasks under normal culture conditions and thus are not independent of tissue culture dispensables. On similar lines US201000330144 provides porous polymeric 3D tubular scaffolds which allow manipulation of pore size, structure and mechanical properties.

The 2D cell culture systems attach to microplate surface in a single monolayer. For any further use of the cells, trypsinization is necessary which comprises the cell number especially for highthroughput needs. US20050054101 discloses a microcarrier suitable for growing cells capable of providing a substrate that will support the growth of cells in culture. The microcarrier is a hydrogel selected from alginate, gelatin, polyacrylamide-copolymerized with collagen or gelatin, polyacrylamide with modified charge or alginate copolymerized with gelatin. A 3D cell culture system using optically transparent microcarrier is particularly advantageous as the cells can be dispensed directly without the need of trypsinization.

Recently, microfluidic cell culture system has been introduced which enables long term perfusion culture of cells in 3D environment. US20090203136 discloses microfluidic cell system that can be used with various standard automated handling systems. One such system described is the integration of microfluidics with a standard 96-well frame that requires no external pumps. Modifications enabling larger gel chambers, automated solution exchange and maintaining long term spatial gradients are also disclosed in the application.

In a different study, Whitesides et al. have reported stacking and destacking layers of paper impregnated with suspensions of cells in extracellular matrix hydrogel (Ref. Paper-supported 3D cell culture for tissue-based bioassays. PNAS (Proceedings of the National Academy of Sciences), 3, 2009, 106:44, 18457-18462). The authors have demonstrated the control and distribution of cultured cells in 3D by fabrication of multilaminate structures of fiber-supported hydrogels composed of chromatography paper impregnated with an extracellular matrix (ECM) hydrogel containing living cells. In this technique, hydrogel precursor is added as a fluid with suspended cells on a paper support which forms gelling at the site. The researchers indicate that it is possible to control oxygen and nutrient gradients in 3D and to analyze the molecular and genetic responses.

Yet another variation is a system marketed by RealBio , which supports mixed cell populations in varied microenvironments. This system allows the control of the culture environment such as the nutrient and gas gradients across the cultured cells by decoupling the supply of nutrient media and metabolic gases.

US20100048411 discloses a cell culture scaffold comprising a polymerized high internal phase emulsion polymer adapted for use in routine tissue culture wells or flasks for analysis of proliferation, differentiation and function of cells. This system however serves as an adjunct to the culturing of cells in tissue culture plates or flasks and does not do away with the traditional culturing methods. QGel™, a trademarked product of QGel Bio envisages a synthetic hydrogel as matrix for 3D cell culture which has protease- sensitive sites and cell adhesion ligands as components of its extracellular matrix.

All the methods of developing 3D cell culture system as described above involve growing 3D cells using polymeric gels, hydrogels, suspension fluid, bead, scaffold, porous sponge, hanging drop and so on. Most of these techniques are complicated, involve robotics and thereby increase the cost factor considerably. In certain cases the cells need to be grown by conventional methods in tissue culture flasks or plates using copious amounts of calf serum increasing the cost of high throughput tissue culture requirements for drug screening. Some of the disadvantages of the prevailing 3D cell culture systems are highlighted below:

Size limitation: The transition of 2D cell cultures using conventional flat surfaces, environment, matrix, and other forms using polymeric and/or non polymeric materials into 3D cell cultures is limited by the smaller size formation of the scaffolds (typically in micron size).

Selection of cell lines: The reported techniques of 3D cell culture system are selective to specific cell lines and it is difficult to grow resistant cell lines; for example MDA MB 231, a triple negative breast cancer cell line does not translate into 3D scaffolds satisfactorily as depicted in Figure 1 wherein comparative growth of MDA-MB-231 breast cancer cells in BD Madtrigel™ matrix (appearing on the left) versus Gel trex™ matrix (appearing on the right) is shown.

Time and volume: One of the most essential properties of a 3D cell culture would be rapid growth of such scaffolds generated using minimal volume of culture media. Thus, there is a need to enhance the surface area or the size of the 3D scaffolds and mimicking the genotype or phenotype traits as in Xenograft models. The versatility of growing the 2D monolayer system into 3D scaffolds even as co- cultures will be of immense significance, for example, as a research tool in drug discovery.

Herein, the applicant discloses a hollow microsphere made of biodegradable polymer that is used to generate 3D cell scaffolds and thus, provides a tool for 3D cell and/or tissue production and evaluate efficacy of cells in in vitro and ex vivo disease models. The 3D cell scaffolds are generated at lower cost and have the advantages of:

• Avoiding the use of tissue culture plates or flasks or vessels; Avoiding excessive use of calf serum as growth promoters in the cell cultures;

Achieving enhanced dimensions such as surface area, size (in millimeter) of the scaffolds;

Effectively growing mixed cell populations concurrently;

Storing the microspheres with the desired cell type thus giving the flexibility to temporarily cease any experimental procedure; and

Successful revival of stored microspheres containing desired cell type.

Summary of the Invention

The present application provides an engineered cell culture matrix suitable for growing cells in hollow polymer microspheres having the ability to transform two- dimensional cells into three-dimensional scaffolds.

In one aspect the cells grown in the hollow polymer microsphere are selected from: epithelial cells, myeloid cells and endothelial cells.

In another aspect, the hollow polymer microsphere comprises multicell cultures grown concurrently in two different layers.

In other aspects of the invention there are processes for preparing an engineered cell culture matrix comprising hollow polymer microspheres.

In a further aspect of the invention, there is a process for screening or testing chemicals, therapeutic agents, differences in temperature, pressure, pH, etc. by growing cells in an engineered cell culture matrix comprising hollow polymer microspheres, treating the microspheres, determining the effects of the chemicals, therapeutic agents, differences in temperature, pressure, pH, etc. on the cells in the microspheres. In a further aspect, the invention also provides a process of screening anticancer therapeutic agents by growing cells in an engineered cell culture matrix comprising hollow polymer microspheres, treating the microspheres with an anticancer therapeutic agent, determining the effects of the anticancer therapeutic agents on growth of the cells in the microspheres and testing the cells for differentiation and/or proliferation.

The invention also provides a process of retrieving two-dimensional monolayers from three-dimensional stage.

Furthermore, in yet another aspect, the hollow polymer microspheres containing the cells are frozen in liquid nitrogen and revived into two-dimensional monolayers.

Brief description of drawings

Figure 1: shows comparative growth of MDA-MB-231 breast cancer cells in BD Madtrigel™ matrix (figure appearing on the left) versus Geltrex™ matrix (figure appearing on the right). Figure 2: depicts polymer matrix containing glass capillary and hollow polymer matrix both containing cells.

Figure 3A: shows cells grown in hollow polymer microspheres. Figure 3B: shows progressive increase of cell number on days 3, 5 and 7 of culture in microspheres.

Figure 4: shows size of cells grown in matrix ranging from 2-3mm. Figure 5: shows head on comparison of cells grown in glass capillary tube versus microsphere, in identical environment. Figure 6: shows concurrent growth of two cell population in microsphere. A549 cells are seen as the outer layer and MCG7-GFP cells as the inner layer.

Figure 7: shows the retrieval sequence of events of MCF7-GFP and A549 cells grown concurrently.

Figure 8A: shows initiation of regeneration of cells in 2S from microsphere

Figure 8B: shows progressive growth of 2D cells as monolayers.

Figure 9: shows in vitro cytotoxicity evaluation in A549 cells grown in

microspheres. P276-00 is the anticancer molecule used in the study.

Figure 10: shows Z-stack images of 3D microsphere system treated with 3 μΜ doxorubicin.

Figure 11: shows time kinetics and cytotoxicity effect of doxorubicin treatment (1-3μΜ) on HL460 derived-3D microsphere system.

Figure 12: shows cytotoxic and cytostatic effect of anticancer compounds on HL460 derived-3D microsphere system.

Figure 13: shows cytotoxic effect of doxorubicin (0.03 - 3 μΜ) in HL460 cells in 2D monolayers versus that observed in HL460 derived-3D microsphere system.

Figure 14: shows comparison of cytotoxic effect of anticancer agents in HL460 derived 3D microsphere system.

Figure 15: shows comparison of cytotoxic effect of anticancer compounds in in 2D monolayer and 3D microsphere system using MCF7 (Breast cancer) cell line. For purposes of the following detailed description, it is to be understood that the invention may assume various alternative variations and step sequences, except where expressly specified to the contrary. Moreover, other than in any operating examples, or where otherwise indicated, all numbers expressing, for example, quantities of ingredients used in the application are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard variation found in their respective testing measurements.

Also, it should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, a range of" 1 to 10" is intended to include all sub-ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10.

In this application, the use of the singular includes the plural and plural encompasses singular, unless specifically stated otherwise. In addition, in this application, the use of "or" means "and/or" unless specifically stated otherwise, even though "and/or" may be explicitly used in certain instances. Detailed Description Of The Invention

Primary cells of mammalian origin are used for toxicity studies or drug screening assays and these include fibroblasts, endothelial cells, epithelial cells and hepatocytes. Mammalian cell cultures are routinely used in drug screening to identify a lead therapeutic and for evaluating the therapeutic through a battery of in vitro tests prior to undertaking animal (in vivo) toxicity and efficacy studies. Thus, improved cell culture systems that mimic the physiological and functional aspects of the cells in order to test cell proliferation and differentiation during drug screening are highly valuable. For this reason cells that exist as multilayers and exhibit three-dimensional architecture are more preferred systems for these type of studies.

The present invention relates to an engineered cell culture matrix comprising hollow polymer microsphere and a method of generating three- dimensional cell scaffold that has a utility as a drug discovery tool in:

(a) generating three-dimensional cell culture and/or tissue

production; and/or

(b) evaluating the efficacy of potential therapeutics in vitro and in ex vivo models.

In one aspect, the present invention is an engineered cell culture matrix for growing cells in hollow polymer microspheres to form a three-dimensional (3D) scaffold.

In one aspect of the invention there is a process of preparing an engineered cell culture matrix comprising hollow polymer microspheres the process comprises the steps of:

(a) preparing a gel by adding a biodegradable polymer to water;

(b) heating the gel to 85°C - 90°C to ensure complete

hydration to obtain a viscous polymer gel; and (c) forming of hollow microspheres by dropping the viscous polymer gel directly into a 2-5% calcium chloride solution. In an embodiment of the invention a 3% calcium chloride solution is used. As alternative, Mg, Cu, and other inorganic ions can be used.

In another aspect of the invention provides a process for preparing an engineered cell culture matrix comprising hollow polymer microspheres the process comprises the steps of:

(a) preparing a gel by adding a biodegradable polymer to water;

(b) heating the gel to 85 °C - 90°C to ensure complete hydration to obtain polymer hydrogel; and

(c) forming hollow microspheres either by trapping air in

polymer hydrogel or by injecting air inside the preformed polymer spheres. In specific an embodiment, the biodegradable polymer is gellan polymer.

GELRITE™, CP Kelco, U.S. Inc., Atlanta, Georgia, USA is a gellan polymer. Generally the amount of Gellan polymer used is 2-3w/v%

The gel formed from Gellan polymer in water is in fluid state. When dropped in solution of calcium chloride ions, the gel forms a solid spherical microsphere. Thereafter, a vacuum is induced in this solid sphere of Gellan, by infusion of an air using syringe. The process describes the method of preparation of soft/elastic spheres from Gellan polymer. A hollow space inside the solid polymer sphere is created by injecting air. Once the air is injected, the space is created, wherein cells in medium are injected. The utility of this space with cells and medium across in equilibrium state, translated 2D cells into 3-D scaffolds mimicking ex vivo scaffolds of cancer cells. In drug discovery set ups, the mass production of such system will be at least semi automated.

The hollow polymer microspheres of the invention thus formed are approximately 5mm to 25mm in diameter.

Following preparation of the microspheres, cells in fetal calf serum (FCS) medium or other media are injected into the hollow of the microsphere. These microspheres can be transferred into 96 well plates having excess FCS medium or other media. Solvent equilibrium is achieved in few minutes and cells are enriched continuously with FCS and/or other nutrients. The fresh medium is replaced every 24 hrs/ or microsphere is transferred into a new well having fresh medium. The microsphere with cultures cells may be maintained in an incubator. Later, fully grown cell scaffolds can be stored in liquid nitrogen at least for 4 weeks and retrieved simply by inserting microspheres with 3D scaffolds into fresh FCS or other suitable medium.

In an aspect of the invention, the scaffold supports the growth of more than one cell type grown simultaneously or concurrently. Two different layers of cells concurrently grown in the microsphere are selected from: epithelial-epithelial cells, epithelial-endothelial cells, epithelial-myeloid cells, endothelial-myeloid cells, endothelial- endothelial cells and myeloid-myeloid cells. In further aspect, the invention demonstrates growing cancer cells derived from epithelial cells, myeloid cells or endothelial cells in 3D multi-spheroid cell cultures. It is envisaged that depending on the combination of cell types grown concurrently, specific cell types will occupy the inner layer of cells and the second the outer layer of cells.

The cells of epithelial origin that are grown in microspheres of the present invention include but are not limited to MCF-7 (breast cancer cells), MDA MB 231 (triple negative breast cancer cells), PC3 (prostrate cancer cells), HL460 (lung cancer cells), Colo205 (colon cancer cells), HCT116 (colon cancer cells), Ovcar (ovarian cancer cells), Pane 1 (pancreatic cancer cells) and A549 (lung cancer cells). Myeloid cells such as the K562 (human bcr-abl leukemia cells), Ba/F3 (mouse monocytic myeloid cells), HL60 (human promyelocytic leukemia cells), THP1 (human acute monocytic leukemia cells), Jurkat (human T cell lymphoblast-like cells), U937 (human histiocytic lymphoma cells), SUP-B 15 (Acute lymphoblastic leukemia cells) and like; and endothelial cells including HUVEC (human umbilical vein endothelial cells) are other examples of cells that can be grown in this three- dimensional cell culture system. The three dimensional cells grown in hollow polymer microspheres can be exposed to chemicals, therapeutic agents, differences is temperature, pressure, pH, etc. to test for, for example, differentiation and proliferation. In a non-limiting aspect, three- dimensional cell matrices of myeloid cells are treated with anticancer therapeutic agents and tested for differentiation and proliferation.

In another aspect, the present invention provides a process for retrieving cells as two-dimensional monolayers from cells grown as three-dimensional scaffolds in the hollow polymer microspheres; wherein said process comprises the steps of:

1 growing the cells in two dimensional monolayers as the first step;

2. injecting the two dimensional cells into the hollow polymer microspheres as the second step;

3 transformation of the two dimensional cells to three- dimensional scaffolds in the microspheres and growing least 7-10 days as the third step;

4. retrieval of two dimensional cell monolayer from the three- dimensional scaffolds by releasing them from the microspheres into a tissue culture medium in a plate or flask as the fourth step. Once the growth is achieved (2 days to 18 days) microspheres can be cut open to separate the multilayered scaffold/ tissue being formed. Thus, the growth of cells in the microspheres does not alter the ability of the cells to grow back in a two dimensional system. The tissue culture environment may be the one that is routinely used in the art.

In an embodiment, the present invention provides a process of storing and revival of three-dimensional cells grown in a hollow polymer microsphere, wherein said process comprises the steps of:

1. freezing the microsphere containing cells in liquid nitrogen; 2. reviving the cells subsequently by thawing; and

3. growing the thawed cells at a later time point as two- dimensional monolayer using tissue culture methods.

The revival time point can be any time following the freezing of cells, for example, 10, 15, 20 or 30 days or even more up to 60 days after the hollow microspheres are frozen. The frozen microspheres are revived into perfectly normal two-dimensional monolayer of cells and thus serve as an efficient tool to scale up cells for high throughput use. In addition, the ability to store the three-dimensional microspheres and revive them as and when required provides the much needed flexibility for researchers while dealing with primary cell cultures.

The hollow microsphere system of the present invention is used to generate three-dimensional microspheres from various cancer cell lines. These microspheres are further used to study the effect of various chemotherapeutic agents using fully grown three-dimensional cancer cell spheroids. The chemotherapeutic agents are anti-cancer agents which include but are not limited to P276-00, doxorubicin, cisplatin, paclitaxel, camptothecin, olaparib, lapatinib and/or any other known anticancer agents or an investigational anticancer agent such as BEZ235 (2-Methyl-2-[4-[3-methyl- 2-oxo-8-(3-quinolinyl)-2,3-dihydro-lH-imidazo[4,5-c]quinolin-l-yl]phenyl]

propanenitrile). The anticancer agents namely doxorubicin, cisplatin, paclitaxel, lapatinib and camptothecin are commercially available. Olaparib can be prepared by a process disclosed in Drugs of Future 2009, 34(2): 101. The investigational drug, BEZ235 can be prepared by the process described in US Patent No. 7667039. The anticancer agent, P276-00 ((+)-trans-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxy-methyl-l-methyl- pyrrolidin— yl)-chromen-4-one hydrochloride), a CDK inhibitor, can be manufactured by the process described in US7271193 which is incorporated herein by reference. Further, the compound P276-00 can be prepared as described herein.

In another aspect, the present invention provides a process of screening anticancer therapeutic agents comprising:

(a) growing cells in hollow polymer microspheres;

(b) treating the microspheres with the anticancer therapeutic agents; and

(c) testing the cells for differentiation and/or proliferation.

It is also relevant herein to note that, though the invention focuses on the use of the hollow polymer microspheres in the screening of anti-cancer therapeutic agents, it will be appreciated by a person skilled in the art that the said microspheres are useful tools to mimic any in vivo physiological condition and thus, is useful for screening any therapeutic agent. In one embodiment, the applicants have established the anticancer efficacy of

P276-00 using the hollow microspheres of the present invention thus establishing the effective use of the 3D cancer cell spheroids as an efficient ex-vivo model in analyzing anticancer therapeutic agents. Furthermore, the polymer microsphere by itself does not impart any phenotypic and genotypic characteristic to the cells.

Definitions

By two-dimensional (2D) cell cultures, it is meant that cells are grown in conventional tissue culture vessel or any flat surface as a monolayer in flasks, plates or inserts. By three-dimensional (3D) cell culture system, scaffold or matrix, it is meant that cells are grown in a hollow polymer matrix with three-dimensional architecture mimicking the responses of real tissues to drugs or toxins as the case may be.

Tissue culture flasks, plates, inserts or any vessel carrying culture monolayers or used for growing culture monolayers are interchangeably used in this disclosure.

The dimension of the polymer matrix can be altered or changed with respect to shape and thus shape is not a limitation for the microsphere of the invention. For example, the shapes of the microsphere can be oval, square or round. By plurality of hollow microspheres, it is herein meant that the hollow microspheres in a culture scenario exist always as multiples and not singularly.

As used herein, the term "approximately" refers to a range of value of + lmm-4 mm of the specified value of the mean diameter of the hollow polymer microsphere. For example, "approximately 25 mm" would imply "24 mm to 26 mm" or "21 mm to 29 mm".

Tissue culture methods, as used in the present disclosure means that cells are grown in appropriate tissue culture medium with supplements as per standard methods known for the specific cells grown.

Although the invention has been described in detail in the disclosure and the following examples for the purposes of illustration, it is to be understood that such details are solely for that purpose and that variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention. The following Examples are intended to illustrate but not to limit the present invention. Examples

Reference example: Preparation of P276-00 ((+)-trans-2-(2-Chloro- phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-yl)-chromen- 4-one hydrochloride)

Molten pyridine hydrochloride (4.1 g, 35.6 mmol) was added to (+)-trans- 2-(2-chloro-phenyl)-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-yl)-5,7-dimethoxy- chromen-4-one (0.4 g, 0.9 mmol) and heated at 180 °C for 1.5 h. The reaction mixture was cooled to 25 °C, diluted with methanol (10 mL) and basified using sodium carbonate to pH 10. The mixture was filtered and the organic layer was concentrated. The residue was suspended in water (5 mL), stirred for 30 min., filtered and dried to obtain the compound, (+)-trans-2-(2-chloro-phenyl)-5,7-dihydroxy-8-(2- hydroxymethyl-l-methyl- pyrrolidin-3-yl)-chromen-4-one .

Yield: 0.25 g (70 %); IR (KBr): 3422, 3135, 1664, 1623, 1559 cm-1 ;

1H NMR (CDC 13, 300MHz): 8 7.56 (d, 1H), 7.36 (m, 3H), 6.36 (s, 1H), 6.20 (s, 1H), 4.02 (m, 1H), 3.70 (m, 2H), 3.15 (m, 2H), 2.88 (m, 1H), 2.58 (s, 3H), 2.35 (m, 1H), 1.88 (m, 1H); MS (ES+): m/z 402 (M+l);

Analysis: C21H20C1N05 C, 62.24 (62.71); H, 5.07 (4.97); N, 3.60 (3.48); CI, 9.01 (8.83). The compound as obtained above (0.2 g, 0.48 mmol) was suspended in isopropanol (5 mL) and 3.5% HC1 (25 mL) was added. The suspension was heated to get a clear solution. The solution was cooled and solid filtered to obtain the compound, (+)- trans-2-(2-Chlorophenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l-methyl- pyrrolidin-3-yl)- chromen-4-one hydrochloride (P276).

Yield: 0.21 g (97 %); mp: 188 - 192 °C ; [ ]D25 = +21.3° (c = 0.2, methanol); IH NMR (CD30D, 300MHz): δ 7.80 (d, IH), 7.60 (m, 3H), 6.53 (s, IH), 6.37 (s, IH), 4.23 (m, IH), 3.89 (m, 2H), 3.63 (m, IH), 3.59 (dd, IH), 3.38 (m, IH), 2.90 (s, 3H), 2.45 (m, IH), 2.35 (m, IH); MS (ES+): rnlz 402 (M +1)( free base)

Example 1: Preparation of hollow polymer microspheres

A viscous gel was prepared using Gellan polymer (GELRITE™, CP Kelco, U.Ss Inc., Atlanta, Georgia, USA). GELRITE is added to water with continuous stirring and the dispersion is heated to 85-90°C to ensure complete hydration. Generally the amount of Gellan polymer used is 2-3w/v %. Gel translates into softer polymer matrix due to divalent complex being formed by using calcium chloride solution.

The polymer spheres are fabricated by dropping the viscous polymer gel directly into the calcium chloride solution (3% w/v). The polymer forms a strong complex instantaneously by replacing the sodium groups in polymer by divalent Ca²⁺ ions. Furthermore, hollow microspheres are formed either by trapping the air in polymer hydrogel or by injecting the air inside the preformed Gellan spheres using, for example, a syringe.

Example 2: Physical characteristics of cells grown in microsphere matrix

Cell Number

Figure 3A depicts the growth of healthy cells in microspheres. The cell numbers progressively increased on 3, 5 and 7 days of culture. The cell number increased from approximately 3x1 cells on day 3 to about 5x10^ cells on day 7 of culture (Figure 3B). Size

While the size of a cell in a two dimensional culture system is about 100-200 μΜ, the cells grown within the microspheres of the invention attained a maximum size of 2-3mm as shown in Figure 4.

Environment induced cell growth

A comparison between glass capillary and hollow polymer matrix for growing cells was carried out. The glass capillary was placed inside the polymer matrix as head on comparison of 2D cell growth into 3D scaffold as shown in Figure 2. Figure 2 depicts polymer matrix containing glass capillary and hollow polymer matrix both containing cells. Hollow matrix was formed by injecting air (at parallel side of the glass capillary) inside the polymer microsphere. Identical cells lines were injected inside the glass capillary as well in hollow microsphere.

Figure 5 shows comparison of cells grown in glass capillary tube versus microsphere in identical environment.

Example 3: Growing mixed cell populations in microsphere matrix

MCF7 GFP cells were first seeded within the polymer microsphere and allowed to grow for 2-3 days. Once the 3D scaffold was developed with MCF7- GFP cells, A549 cells were inoculated in the same hollow microsphere. Growth progression as well as the pattern was monitored and images were taken for analysis.

Figure 6 depicts the concurrent growth of mixed population of cells in microsphere. In Figure 6, A549 (of epithelial origin) cells are seen as the outer layer and MCF7-GFP (of epithelial origin) cells grown as the inner layer. Both cell layers seem to be perfectly nonnal and healthy, thus reiterating that mixed cell populations can be grown routinely in microspheres thus providing a three-dimensional cell culture system for different cell types grown concurrently.

Example 4: Retrieval of cells to 2D system from 3D system The primary cells were firstly grown as a two dimensional monolayer in appropriate tissue culture conditions. For example, A549 (lung carcinoma) monolayers were grown in tissue culture medium (RPMI with FCS (Fetal calf serum)). Once healthy, they were injected into the hollow microspheres and were found to translate into three- dimensional scaffolds. After 7-10 days, the microsphere with the cells were either stored and revived (as shown in Example 5) or were retrieved back into the two dimensional state by releasing them from the microspheres into appropriate tissue culture medium.

Figure 7 shows the retrieval sequence of events of MCF7-GFP and A549 cells grown concurrently. First, 3D scaffold was developed using MCF7-GFP cells. Later, A549 cells were injected / inoculated in the same hollow microsphere having MCF- GFP scaffold developed. A549 cells initiates to grow on earlier scaffold (i.e. MCF- GFP). Example 5: Storage and revival of cells grown in hollow microspheres

The microspheres containing cells (MCF7 and A549 cells grown independently) were frozen or stored in liquid nitrogen. The cells were revived post 5, 10, 15, 20 or 30 days of storage and grown as a monolayer using tissue culture revival and growing techniques.

Figure 8A and 8B show the initiation of revival and the progressive growth of MCF7 and A549 cells in monolayer respectively. No significant phenotypic change was observed in cells revived/retrieved from

3D scaffolds to 2D in comparison to cells growing in 2D monolayer system. Example 6: Response of cells grown in microspheres to chemotherapeutic agent P276-00

The microspheres containing the cells were allowed to grow as 3D cultures. After 3 days, chemotherapeutic agent P276-00 was added to the culture medium at varying concentrations (3 μΜ and 10 μΜ) and its effect on the growth of 3D cultures was analyzed. After 48 hrs of treatment with P276-00, microspheres were visualized

under microscope for studying the growth pattern of the cultures. Images of control versus the treated microspheres were captured.

It was observed that treatment with P276-00 induced cytotoxicity in a dose- dependent manner, as demonstrated through disruption in the formation and growth of 3D cultures within the microspheres.

Figure 9 provides the therapeutic potential of P276-00 against A549 cells grown in microspheres.

Example 7: Evaluation of penetration of doxorubicin in the 3D microsphere system

Penetration of a drug such as doxorubicin in the 3D microsphere system was evaluated. Z-stack of 3D HL460 derived microsphere system w a s treated with 3 μΜ doxorubicin for 18 hrs. Figure 10 provides Z-stack images of 3D microsphere system treated with 3 μΜ doxorubicin.

Example 8: Cytotoxic effect of doxorubicin on HL460 derived 3D microsphere system

HL460 derived 3D microsphere system w a s treated with doxorubicin (1 -3 μΜ) for 48 hours. It was observed that 1 μΜ doxorubicin inhibited the cell growth and showed greater cytotoxicity within 48 hrs, whereas 3μΜ doxorubicin demonstrated cytotoxicity effect within 24 hr.

Figure 11 shows time kinetics and cytotoxicity effect of doxorubicin treatment (1- 3μΜ) on HL460 derived-3D MCS.

Example 9: Evaluation of effect of BEZ235, laptinib and paclitaxel on HL460 derived-3D microsphere system.

HL460 derived 3D microsphere system w as treated with BEZ235 (1 μΜ), lapatinib (1 μΜ) and paclitaxel (1 μΜ). It was observed that BEZ235 (1 μΜ) and lapatinib (1 μΜ) showed cytostatic effect, while paclitaxel (1 μΜ) exhibited cytotoxic effect on HL460 derived 3D microsphere within 48 hr of incubation.

Figure 12 shows cytotoxic and cytostatic effect of anticancer compounds on HL460 derived-3D microsphere system.

Example 10: Dose dependent effect of doxorubicin in 2D monolayer culture versus 3D microsphere system

The percentage cytotoxic effect using H460 cells for doxorubicin (0.03 - 3 μΜ) in 2D monolayers in 48 hrs was compared with HL460 derived-3D microsphere system treated with doxorubicin (0.03 to 3 uM) with exposure of 96 hrs.

Figure 13 depicts cytotoxic effect of doxorubicin (0.03 - 3 μΜ) in HL460 cells in 2D monolayers for 48 hrs versus that observed in HL460 derived-3D microsphere system with exposure of 96 hrs.

Example 11: Comparison of cytotoxic effect of anticancer agents in HL460 derived 3D microsphere system

HL460 derived 3D microsphere system w as treated with anticancer agents such as doxorubicin, cisplatin, BEZ235, olaparib, paclitaxel, lapatinib and P276. The anticancer agents were evaluated in concentration ranging from 1 -10 μΜ. The results of this study are presented in the following table.

Figure 14 depicts comparison of cytotoxic effect of anticancer agents in HL460 derived 3D microsphere system

Example 12: Comparative percentage cytotoxicity profile of anticancer agents in 2D monolayer and 3D microsphere system using MCF7 (Breast cancer) cell line. The percentage cytotoxic effect using MCF7 (Breast cancer) cell line for certain anticancer agents at concentration at different concentration in 2D monolayers in 48 hrs was compared with MCF7 (Breast cancer) cell derived-3D microsphere system for 96 hrs. Figure 15 depicts comparison of cytotoxic effect of anticancer compounds in 2D monolayer and 3D microsphere system using MCF7 (Breast cancer) cell line.

Claims

We claim:

1. An engineered cell culture matrix suitable for growing cells comprising a plurality of hollow polymer microspheres having the ability to translate or transform two- dimensional cells into three-dimensional scaffolds.

2. The engineered cell culture matrix as claimed in claim 1 , wherein the hollow polymer microspheres have a mean diameter of approximately 5 mm to 25 mm.

3. The engineered cell culture matrix as claimed in claim 1, wherein the cells grown are selected from epithelial cells, myeloid cells and endothelial cells.

4. The engineered cell culture matrix as claimed in claim 3, wherein the epithelial cells are selected from: MCF-7, MDA MB 231, PC3, MCF-7, MDA MB 231 , PC3, HL460, Colo205, HCT116, Ovcar, Pane 1 and A549.

5. The engineered cell culture matrix as claimed in claim 3, wherein the myeloid cells are selected from: K562, Ba/F3, HL60, THP1, Jurkat, U937 and SupB 15.

6. The engineered cell culture matrix as claimed in claim 3, wherein the endothelial cells are HUVEC.

7. The engineered cell culture matrix as claimed in claim 1, wherein the microsphere comprises multi-cell culture grown concurrently in two different layers.

8. The engineered cell culture matrix as claimed in claim 7, wherein the two different layers are selected from: epithelial-epithelial cells, epithelial- endothelial cells, epithelial-myeloid cells, endothelial-myeloid cells, endothelial- endothelial cells and myeloid- myeloid cells.

9. The engineered cell culture matrix as claimed in claim 7, wherein the microsphere comprises an inner layer of MCF-7 cells and an outer layer of A549 lung carcinoma cells.

10. A process of preparing hollow polymer microspheres comprising the steps of:

(a) preparing a gel by adding a biodegradable polymer to water;

(b) heating the gel to 85 °C - 90°C to ensure complete

hydration to obtain viscous polymer hydrogel; and

(c) formation of hollow microspheres by dropping the viscous

polymer gel directly into 3% calcium chloride solution.

11. A process for preparing hollow polymer microspheres comprising the steps of:

(a) preparing a gel by adding a biodegradable polymer to water;

(b) heating the gel to 85°C - 90°C to ensure complete hydration to obtain polymer hydrogel; and

(c) forming hollow microspheres either by trapping air in the

polymer hydrogel or by injecting air inside the preformed polymer spheres.

A process of screening anticancer agents comprising:

(a) growing cells in hollow polymer microspheres;

(b) treating the microspheres with the anticancer agents;

and

(c) testing the cells for differentiation and/or proliferation.

13. The process as claimed in claim 12, wherein the anticancer agent is selected from: P276-00, doxorubicin, cisplatin, paclitaxel, camptothecin, olaparib, lapatinib or BEZ235.

14. A process for retrieving cells as two-dimensional monolayers from cells grown as three-dimensional scaffolds in the hollow polymer microspheres; wherein said process comprises the steps of:

a) growing the cells in two dimensional monolayers as the first step; b) injecting the two dimensional cells into the hollow polymer microspheres as the second step;

c) transformation of the two dimensional cells to three- dimensional scaffolds in the microspheres and growing for at least 7-10 days as the third step; and

d) retrieving two dimensional cell monolayer from the three- dimensional scaffolds by releasing them from the microspheres into a tissue culture medium in a plate or flask as the fourth step.

15. A process of storing and revival of three-dimensional cells grown in a hollow polymer microsphere comprising the steps of:

a) freezing the microsphere containing cells in liquid nitrogen; b) reviving the cells subsequently by thawing; and c) growing the thawed cells at a later time point as two-dimensional monolayer using a tissue culture method.