BIOMIMETIC AMNIOTIC MEMBRANE NICHE FOR STEM CELLS

Abstract

In this invention we propose a method to compose a stem cell culture niche platform, which is based on the use of the human amniotic membrane. Fluid dynamic, mechanical and topographic factors are additionally included in this niche to provide various factors essential for achieving an enhanced biomimetic microenvironment of the cultured stem cells. The amniotic membrane is mounted into various types of culture platforms to suit a wide range of research applications. The rich composition of the membrane with anti-inflammatory, anti microbial, matrix and adhesion molecules in addition to various growth factors suits its application as a complex biomimetic material. The platform includes micro channels to allow continuous exchange of media and creates a dynamic flow of the fluid surrounding the cells in an attempt to simulate the in vivo conditions in which the stem cell typically reaches its ideal proliferation, expansion or differentiation. The method disclosed herein supports a wide range of applications in stem cell research such as the investigation of the optimal conditions for stem cell culture and the effect of various medications and external factors. It can be also applied in investigating the effect of the amniotic membrane and the mechanical factors on the behavior of stem cells and cancer stem cells. Another model of the niche is proposed as an in vivo moldable and implantable carrier for delivering stem cell based therapies in a wide range of diseases especially those associated with aging or decline of specialized cell function such as diabetes, cardiovascular, neurological, hormonal, renal and liver disorders, cancer, and diseases associated with inflammation and disordered immunity. Furthermore, the lack of HLA molecules renders the membrane nave to minimize rejection, which could be valuable for transplantation purposes.

Claims

1. A method of creating one or more biomimetic cell culture platform to mimic a native cell niche including the use of biological membranes and tissues embedded in three dimensional surface with a fluid dynamic mechanical factor.

2. As mentioned in claim #1, the platform could be of various shapes and sizes including flasks, plates, dishes, wells, dishes, channels, vessels, capsules, discs or any custom made containers

3. As mentioned in claim #1, the biomimetic platform of claim 1 is fabricated of Polydimethylsiloxane (PDMS), silicone or other flexible polymers and elastomers.

4. The biological membranes and tissue mentioned in claim 1, wherein human amniotic membrane, umbilical cord and placenta can be included in the platform.

5. The amniotic membrane mentioned in claim 4, wherein prior preparation of the membrane surface is done using chemical and mechanical methods to produce fully decellularized or partially decellularized surfaces based on application.

6. The three dimensional surface mentioned in claim 1, wherein patterns on the lining surface of the culture platform are made to mimic the microarchitecture and topography of the native in vivo cell niche.

7. The three dimensional surface mentioned in claim 1, wherein amniotic membrane based nano fibers are patterned in a network or aligned in various distribution to comprise the topography specific for the differentiating cells of interest.

8. The three dimensional surface mentioned in claim 1, wherein the surface topography can be patterned using three dimensional printers either for the prototype original material or for the amniotic membrane surface.

9. The three-dimensional surface mentioned in claim 1, wherein a free hanging piece can be inserted as a three dimensional disc for cell expansion on both sides.

10. The free hanging piece mentioned in claim 9, wherein the three dimensional surface is composed by the amniotic membrane and further enhanced by molding the amniotic membrane on configurations or arrays printed on the hanging piece.

11. As mentioned in claim #1, the cultured cells comprise proliferating, expanded or differentiated stem cells.

12. The stem cells mentioned in claim 11, comprising embryonic stem cells, adult stem cells, perinatal stem cells, fetal stem cells, cancer stem cells, induced pluripotent stem cells or any of their other subtypes.

13. The fluid dynamic factor mentioned in claim 1, comprising a method to create a continuously flowing or exchanged fluid surrounding the growing or differentiating cells.

14. The fluid mentioned in claim 13, wherein the fluid can be a culture medium, serum preferably human or cord blood derived, activated rich plasma or amniotic fluid.

15. The fluid dynamics mentioned in claim 1, wherein delivery of the fluid is through one or more inlet and outlet channels.

16. The inlet or outlet channels in claim 15, wherein channels can be stand free independent channels or can be microchannels of a microchip.

17. The outlet channels mentioned in claim 15, wherein such channels are equipped with one or more various size filters to prevent blocking of the channel by cells and to maintain a continuous flow.

18. The filters mentioned in claim 17, wherein filters can be external filters connected to the platform or internal built in posts inside micro channels.

19. The fluid dynamic factor mentioned in claim 1, wherein an external, portable or implantable pump system can be attached to the platform channels to automate the process of fluid exchange and suits in vivo stem cell applications.

20. The biomimetic platform mentioned in claim 1, wherein various cell culture platforms are used including microchips and lab-on-chip techniques.

21. The microchips mentioned in claim 20, wherein channel microfluidics platform or digital microfluidics platform is employed.

22. The microchips in platform mentioned in claim 20, wherein chips are made of modified and unmodified surfaces.

23. The biomimetic platform mentioned in claim 1, wherein the proposed platform can be used in stem cell research studies for a wide range of medical diseases.

24. The medical diseases mentioned in claim 23, wherein any medical dysfunction or disease model can be studied in particular cardiovascular diseases, neurological diseases, spinal cord injury, infections, cancer, diabetes, inflammatory, hormonal and immune disorders.

25. The biomimetic platform mentioned in claim 1, wherein an in vivo implantable model can be made by modifications of the research compatible version to suit therapeutic application.

26. The in vivo platform mentioned in claim 25, wherein stem cell based therapies can be delivered due to the low immunogenic properties of the amniotic membrane that support its use in transplantation therapies.

27. The in vivo platform mentioned in claim 25, wherein the human amniotic membrane is skeletonized in various shapes to create a cell carrier that can deliver undifferentiated or differentiated stem cells in vivo.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Preferred embodiments of the invention will now be described, by way of example only, with reference to the drawings, in which:

[0047] FIG. 1: It shows a drawing of one example of a design of the platform 1, lined by the human amniotic membrane 2, and one internal knob 3 representing said a mobile plate covered with amniotic membrane and has a three dimensional surface topography. Fluid inflow and outflow are shown 7, external connecting tubes for inflow and outflow channels are shown 4 and 5. An external source for inducing mobility 6 could be included to support the internal flow dynamics or to replace it based on the application

[0048] FIG. 2: Shows a drawing of another example of the design of the platform wherein, 12 denotes the provision of repeated units of the same platform as required by the application.

[0049] FIG. 3: Shows a drawing of cut section of a spherical design of the platform that contains an internal spherical porous knob also covered by the membrane. The fluid delivering channel 9 allows the delivery of culture media and other important constituents and is connected through the internal side of the knob. Filters are added to the fluid channels to prevent the exit of cells during the flow.

[0050] FIG. 4: Shows a drawing of an implantable system for therapeutic application and stem cell transplantation. It is composed of an external amniotic based skeleton 13 surrounding and patterned on harder posts or frame 14 to mold it for the desired shape. It can connect to the in vivo microenvironment through micro channels 16, equipped with internal built in micro filters of various sizes 15 based on the application of interest basically to either allow the exit or exchange of cells and molecules or to allow the exit of molecules and factors secreted by those cells without the passage of cells.

[0051] FIG. 5: This figure shows various prototypes that are in current development to investigate the effectiveness of the platform. P1 is for a platform that employs a regular petri dish. P2 for 6-well plate lined with amniotic membrane, P3 shows successive steps of developing a distinct prototype that enables easier incorporation of surface and dynamic factors (a-d). P4 is for a prototype that is dependent on a microchip reservoir connected with micro channels (inlet and outlet microchannels fabricated inside the chip).

[0052] FIG. 6: This figure shows a surface electron microscope image of the human amniotic membrane after decellularization. The natural three-dimensional topography is well demonstrated at 5000 magnification S1. An image J software used to further highlight this topography using an inverse surface mode S2 and a 3D surface plot function of the same image S3.

[0053] FIG. 7: This figure shows preliminary data of human umbilical cord blood mononuclear cells that was isolated and cultured on the amniotic membrane lined plates. The proliferation of cells on successive days of the culture is shown as D0, D3, 4 and 5.

[0054] The examples and embodiments described herein are for illustrative purposes only. Modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application. The figures are not to scale and some features may be exaggerated or minimized to show details of particular elements while related elements may have been eliminated to prevent obscuring novel aspects. Accordingly, specific structural and functional details disclosed herein are not to be interpreted as restrictive but as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention. As used herein, the terms about, and approximately when used in conjunction with ranges of concentrations, temperatures or other physical or chemical properties or characteristics is meant to cover slight variations that may exist in the upper and lower limits of the ranges of properties/characteristics. Also, as used herein, the terms comprises, comprising, includes and including are to be construed as being inclusive and open ended, and not exclusive. Specifically, when used in this specification including claims, the terms comprises, comprising, includes and including and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components. The foregoing description of the preferred embodiments of the invention has been presented to illustrate the principles of the invention and not to limit the invention to the particular embodiment illustrated. It is intended that the scope of the invention be defined by all of the embodiments encompassed within the following claims and their equivalents.