MULTILAYER SHEET OF CELLS

Abstract

A multilayer sheet of cells, comprising a layer of endothelial cells, and a layer of podocytes is disclosed.

Claims

1. A multilayer sheet of cells, comprising a layer of endothelial cells, and an adjacent layer of podocytes.

2. The multilayer sheet of cells according to claim 1, wherein the endothelial cells are glomerular endothelial cells.

3. The multilayer sheet of cells according to claim 1 or claim 2, further comprising an extracellular matrix disposed between the cell layers.

4. The multilayer sheet of cells according to any one of claims 1 to 3, wherein the multilayer sheet of cells is provided on a support surface.

5. The multilayer sheet of cells according to any one of claims 1 to 4, wherein the multilayer sheet of cells is detachable from the support surface without enzymatic treatment, optionally wherein the support surface is a temperature responsive support surface.

6. The multilayer sheet of cells according to any one of claims 1 to 5, wherein the cell layers are confluent cell layers.

7. The multilayer sheet of cells according to any one of claims 1 to 6, which is tolerant of flow conditions.

8. The multilayer sheet of cells according to any one of claims 1 to 6, which is impervious to proteins larger than about 100 kDa.

9. An in vitro cell culture, comprising a layer of endothelial cells, and an adjacent layer of podocytes, optionally wherein the endothelial cells are glomerular endothelial cells.

10. An in vitro cell culture according to claim 9, comprising an extracellular matrix disposed between the cell layers.

11. An in vitro cell culture according to claim 9 or claim 10, provided on a support surface.

12. An in vitro cell culture according to claim 11, wherein the support surface is a surface from which the multilayer sheet of cells is detachable without enzymatic treatment, optionally wherein the support surface is a temperature responsive support surface.

13. An in vitro cell culture according to any one of claims 9 to 12, wherein the cell layers are confluent cell layers.

14. A device, having two compartments separated by a multilayer sheet of cells according to any one of claims 1 to 8.

15. The device according to claim 15, additionally comprising a liquid/fluid permeable support for the multilayer sheet of cells.

16. The device according to claim 14 or claim 15, comprising a liquid/fluid inlet to the compartment adjacent the layer of endothelial cells, and a liquid/fluid outlet from the compartment adjacent the layer of podocytes.

17. The device according to claim any one of claims 14 to 16, additionally comprising a compartment comprising renal epithelial cells.

18. The device according to claim any one of claims 14 to 17, wherein the compartment comprising renal epithelial cells is in liquid/fluid communication with the compartment adjacent the layer of podocytes.

19. Use of a multilayer sheet of cells according to any one of claims 1 to 8, or of a device according to any one of claims 14 to 18, in an in vitro assay.

20. The use according to claim 19, wherein the assay is a toxicity assay.

21. The use according to claim 19 or claim 20, wherein the assay measures transfer of a substance across the multilayer sheet of cells.

22. The use according to any one of claims 19 to 21, wherein the assay measures a property of the multilayer sheet of cells, or a property of a component of the multilayer sheet of cells.

23. An artificial kidney, comprising a multilayer sheet of cells according to any one of claims 1 to 8, or a device according to any one of claims 14 to 18.

24. A dialysis apparatus, comprising a multilayer sheet of cells according to any one of claims 1 to 8, or a device according to any one of claims 14 to 18.

25. Use of a multilayer sheet of cells according to any one of claims 1 to 8, a device according to any one of claims 14 to 18, an artificial kidney according to claim 23, or a dialysis apparatus according to claim 24, to filter blood or plasma.

26. The multilayer sheet of cells according to any one of claims 1 to 8, device according to any one of claims 14 to 18, artificial kidney according to claim 23, or dialysis apparatus according to claim 24, for use in a method of treatment by dialysis.

27. A method of treating a patient in need of dialysis, the method comprising use of a multilayer sheet of cells according to any one of claims 1 to 8, a device according to any one of claims 14 to 18, an artificial kidney according to claim 23, or a dialysis apparatus according to claim 24, to dialyse blood or plasma.

28. A method for producing a multilayer sheet of cells, the method comprising: (i) providing a layer of endothelial cells; (ii) providing a layer of podocytes; and (iii) contacting the layers of cells and culturing the contacted cell layers in vitro; thereby producing a multilayer sheet of cells.

29. A method for producing a multilayer sheet of cells, the method comprising: (i) providing a layer of endothelial cells, optionally glomerular endothelial cells; (ii) layering podocytes onto the layer of endothelial cells; and (iii) culturing the cells in vitro; thereby producing a multilayer sheet of cells.

30. The method according to claim 29, wherein step (i) comprises contacting endothelial cells or glomerular endothelial cells with a support surface, and culturing the cells on the support surface in vitro.

31. The method according to claim 30, wherein the support surface is a surface from which the multilayer sheet of cells is detachable without enzymatic treatment, optionally wherein the support surface is a temperature responsive support surface.

32. The method according to any one of claims 29 to 31, wherein the method additionally comprises: (iv) detaching the multilayer sheet of cells from the support surface.

33. A method for producing a multilayer sheet of cells, the method comprising: (i) providing a layer of podocyte cells; (ii) providing a layer of endothelial cells, optionally glomerular endothelial cells, (iii) placing the layer of endothelial cells onto the layer of podocyte cells, or placing the layer of podocyte cells onto the layer of endothelial cells; and (iii) culturing the cells in vitro; thereby producing a multilayer sheet of cells.

34. The method according to claim 33, wherein one or both of the layer of podocyte cells of (i) and layer of endothelial cells of (ii) is a layer on a support surface from which the cell layer is detachable without enzymatic treatment, optionally wherein the support surface is a temperature responsive support surface.

35. The method according to claim 33 or 34, wherein the method additionally comprises detaching the multilayer sheet of cells from the support surface.

36. A bilayer sheet of cells, comprising a layer of endothelial cells, and a layer of podocytes.

37. A bilayer sheet of cells consisting of a layer of endothelial cells and a layer of podocytes.

38. A bilayer sheet of cells consisting of a layer of endothelial cells, a layer of podocytes and extracellular matrix disposed between the cell layers.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0234] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures:

[0235] FIG. 1. Photographs showing a sheet of GEnCs (passage 27) on a confluent layer of podocytes (AB passage 20). Anti-PECAM panels show staining of cells with an anti-PECAM antibody. Anti-synaptapodin shows staining of cells with an anti-synaptapodin antibody. Actin shows staining of cells for actin. Merge top right panel shows the merge of the top left and top middle images. Merge bottom right panel shows the merge of the bottom left and bottom middle images.

[0236] FIG. 2. Electron micrographs showing a sheet of glomerular endothelial cells (GEnCs; (passage 31) on a layer of podocytes (GFP-AB passage 15+14). GEnC indicates a glomerular endothelial cell. Aclar indicates aclar.

[0237] FIG. 3. Photographs showing podocytes (AB passage 25) cultured for 24 h under static (top panels) or flow conditions (lower panels) at a flow rate of 500 l/min. Vinculin shows staining of cells for vinculin, Actin shows staining of cells for actin, and Merge shows the merge of vinculin and actin staining images.

[0238] FIG. 4. Photographs showing podocytes (GFP-AB passage 15+10) and GEnC (passage 30) under static conditions (upper two rows of panels) and flow conditions (lower two rows of panels). Dapi indicates staining of the cells with Dapi. GFP indicates GFP fluorescence signal from the cells. PECAM panels show staining of cells with an anti-PECAM antibody. Merge panels show merges of Dapi, GFP and PECAM images for each row of panels.

[0239] FIG. 5. Electron micrographs of podocytes (nephrin-FLAG; passage 18+13) cultured under static conditions (left column panels) and flow conditions (right column panels). Arrows indicate concentrations of actin fibres.

[0240] FIG. 6. Schematic of an exemplary embodiment of a flow chamber (101), divided into two compartments (102, 103) by a bilayer cell sheet (104). The upper compartment (102) has an inlet (105) and an outlet (106) allowing fluid to flow over endothelial cells of the sheet. The lower compartment provides for collection of filtrate and has an outlet (107) allowing filtrate to be removed.

[0241] FIGS. 7A and 7B. Diagram illustrating generation of self-standing cell sheets generated by culturing cells to confluence on temperature sensitive surfaces. (A) Culture on substrate having poly(N-isopropylacrylamid)(NIPAAm) chains grafted to the substrate surface at 37 C. provides for cell attachment. (B) NIPAAm chains become hydrophilic at 21 C. causing them to swell, de-bond the cells and enable release of a cell sheet.

EXAMPLES

[0242] The following examples demonstrate that:

(1) Glomerular endothelial cells (GEnCs) can be grown on thermo-responsive surfaces and lifted off as cell sheets (see Example 1.2)
(2) Podocytes and proximal tubule cells do not grow well on thermo-responsive surfaces (see Examples 1.1 and 1.3)
(3) Podocytes can be grown on a layer of GEnCs (see Examples 1.4 and 2.3)
(4)(i) GEnCs can be cultured to confluence on a temperature responsive surface, (ii) a layer of podocytes can be cultured on top of the GEnC layer to form a bilayer of GEnCs and podocytes, and (iii) the bilayer can be detached from the temperature responsive surface as a bilayer sheet of cells (see Example 1.4).
(5) Podocytes and GEnC have different morphological appearances and distribution of focal adhesion/junctional proteins under flow conditions as compared to the cells grown under static conditions (see Examples 3-5).
(6) Podocytes and glomerular endothelial cells are viable in coculture, under both static and flow conditions (see Examples 4 and 5)

Example 1: Cell Sheets from Temperature-Responsive DishesAnalysis by Light Microscopy

[0243] Cell sheets were prepared on temperature-responsive UpCell Surface cell culture dishes (Thermo Scientific Nunc), which comprises PIPAAm, according to the manufacturer's instructions.

[0244] Briefly, cells were seeded in cell culture media on UpCell Surface cell culture dishes and cultured in vitro to confluence at 37 C. For harvesting the cell layer, the cell culture media was aspirated, and about 0.5 ml of cooled or room temperature cell culture media was added to the cells to reduce the temperature of the cells and culture dishes.

[0245] A transfer membrane was applied to the surface of the cultured cells, and the cultured cells and culture dishes were allowed to cool to provide the temperature responsive dish with a hydrophilic surface.

[0246] Cell sheets were then removed from the UpCell Surface cell culture dishes by lifting up the transfer membrane, with the layer of cultured cells attached to it.

[0247] Where cell sheets were transferred to another layer of cells in cell culture, the sheet of cells attached to the transfer membrane was placed onto a layer of cells in culture. The sheet of cells was allowed to attach to the cells in culture, and additional cell culture media was added to facilitate detachment of the transfer membrane from the bilayer of cells. The transfer membrane was then removed from the culture.

1.1 Podocytes do not Form a Detachable Cell Sheet on Temperature-Responsive (TR) Dishes

[0248] Podocytes do not grow well on polymer on temperature responsive dishes. Cells were very patchy and unhealthy looking after seeding on dishes. Coating the dishes with the glomerular extracellular matrix (ECM) ligand collagen IV did not improve growth of podocytes on temperature responsive dishes.

1.2 Glomerular Endothelial Cells (GEnCs) Grow Well on TR Dishes.

[0249] Glomerular endothelial cells grow well on temperature responsive dishes, and formed a detachable cell sheet after one week at 33 C. (split one from a confluent T75 flask of cells, onto two 3.5 cm dishes).

[0250] Cell sheets were cut and transferred onto confluent layer of podocytes grown on coverslips and aclar. Cells were incubated at 37 C. for 14 days. Subsequently, cocultured cells were stained with synaptopodin (a podocyte marker) and with PECAM1 (a GEnC marker). Both cell types could be detected (FIG. 1).

1.3 Proximal Tubular Epithelial Cells do not Grow Well on TR Dishes

[0251] HK-2 (proximal tubular epithelial cells) do not grow very well on temperature responsive dishes. Cells were very round and most of the cells did not attach to the dish. The manufacturer's recommendation is not to grow these cells to confluence.

1.4 Preparing a Bilayer of a GEnC Sheet of Cells and a Layer of Podocytes on a TR Dish

[0252] GEnCs were cultured in vitro to a confluent monolayer of cells on a temperature responsive dish. Podocytes were subsequently added to the cell culture, and cultured in vitro to provide a confluent monolayer of cells on top of the layer of GEnCs.

[0253] Subsequently, the cells and culture dishes were cooled to provide the temperature responsive dish with a hydrophilic surface for detachment of the bilayer of GEnCs and podocytes.

[0254] Remarkably, the GEnC+podocyte bilayer detached from the temperature responsive dish as a single sheet of the bilayer of cells. The bilayer could be detached from the temperature responsive surfaces using tweezers, and could also be floated-off the surfaces with cell culture media.

Example 2: Analysis of Cell Sheets by Electron Microscopy

[0255] For improved imaging of cultures of GEnC and podocytes, cocultures were imaged grown on aclar and prepared for electron microscopy (EM). Transmission EM was performed using a Tecnai 12 Biotwin.

2.1 GEnC Sheets (Passage 29) Placed on a Layer of Podocytes (GFP-AB Passage 15+10)

[0256] GEnC (passage 29) sheets were placed on a layer of podocytes (GFP-AB passage 15+10), and the cells were differentiated for 14 days. Overall the cells were not healthy, there was evidence of significant cell death, and the podocytes appeared flatter than normal.

2.2 GEnC Sheets (Passage 31) Placed on a Layer of Podocytes (GFP-AB Passage 15+10)

[0257] GEnC (passage 31) sheets were placed on a layer of podocytes (GFP-AB passage 15+14). Cells were left to differentiate for 14 days. There was lots of cell death and podocytes appeared flatter than usual (FIG. 2).

2.3 GEnC Sheets (Passage 31) Differentiated for 7 Days, and Layered with Podocytes (GFP-AB Passage 15+10)

[0258] GEnC (passage 27) were differentiated for 7 days, and then podocytes (GFP-AB passage 15+10) were seeded onto the GEnC layer. The cells were then differentiated for another 7 days. Cells were healthy and separated.

[0259] In view of this result, and the successful production of a bilayer as described in 1.4 above, it was therefore decided that for preparing bilayers of cells, the method would be to culture a layer of glomerular endothelial cells, and to then layer and culture podocytes on top of the layer of glomerular endothelial cells, to produce a bilayer of glomerular endothelial cells and podocytes.

Example 3: Analysis of Podocytes Under Flow Conditions by Light Microscopy

[0260] QuasiVivo flow chambers were used to investigate whether podocytes can tolerate flow conditions. Using the QuasiVivo chambers cells were cultured under static and flow conditions. A variable flow rate was used over a time course. Imaging was performed with a slide scanner.

3.1 Podocytes (AB Passage 24) Cultured for 24 Hours at a Flow Rate of 480 l/Min

[0261] Podocytes (AB passage 24) were cultured under flow conditions at a flow rate of 480 l/min, or under static conditions, for 24 hours. Cells cultured under flow conditions formed a more complete monolayer (i.e. were more confluent) than cells cultured under static conditions seeded at the same density.

3.2 Podocytes (AB Passage 25) Cultured for 24 Hours at a Flow Rate of 500 l/Min

[0262] Podocytes (AB passage 25) were cultured under flow conditions at a flow rate of 500 l/min, or under static conditions, for 24 hours. Cells cultured under flow conditions were more confluent than cells cultured under static conditions seeded at the same density (FIG. 3).

3.3 Podocytes (AB Passage 26) Cultured for 7 Days at a Flow Rate of 450 l/Min

[0263] Podocytes (AB passage 26) were cultured under flow conditions at a flow rate of 450 l/min, or under static conditions, for 7 days. Cells cultured under flow conditions were more confluent than cells cultured under static conditions seeded at the same density, but the difference was less pronounced than at 24 hours. Focal adhesions (FAs) of the podocytes were longer and more abundant for cells cultured under flow conditions, and the localisation of vinculin was primarily to the FAs.

3.4 Podocytes (Nephrin-FLAG, Passage 18+11) Cultured for 5 Days at a Flow Rate of 430 l/min

[0264] Podocytes having stable expression FLAG-tagged nephrin (passage 18+11) were cultured for 5 days at a flow rate of 430 l/min, or under static conditions. Overall nephrin expression was increased in cells cultured under flow conditions relative to cells cultured under static conditions.

3.5 Podocytes (Nephrin-FLAG, Passage 18+13) Cultured for 5 Days at a Flow Rate of 450 l/min

[0265] Podocytes having stable expression FLAG-tagged nephrin (passage 18+13) were cultured for 5 days at a flow rate of 450 l/min, or under static conditions and analysed for surface nephrin expression (cells were not permeabilised prior to imaging).

Example 4: Analysis of GEnCs and Podocytes Under Flow Conditions by Light Microscopy

[0266] QuasiVivo flow chambers were used to investigate whether GEnCs and podocytes can tolerate flow conditions. Using the QuasiVivo chambers cells were cultured under static and flow conditions. A variable flow rate was used over a time course. Imaging was performed with a slide scanner.

4.1 Coculture of Glomerular Endothelial Cells (Passage 31) and Podocytes (Nephrin-FLAG, Passage 18+13)

[0267] Glomerular endothelial cells (passage 31) were grown for 8 days at 37 C. on cover slips. Podocytes (nephrin Flag passage 18+13) were added, and cells were then cultured under flow conditions or static conditions for 5 days. Nephrin expression was increased in cells cultured under flow conditions relative to cells cultured under static conditions.

4.2 Coculture of Glomerular Endothelial Cells (Passage 30) and Podocytes (GFP-AB, Passage 15+10)

[0268] Three QuasiVivo flow chambers were used in this flow experiment. One chamber for podocytes, one chamber for endothelial cells and one chamber for both cell types together. The flow rate was 540 l/min and the same number of cells were used for the static control. Cells were incubated for 5 days at 37 C. under static or flow conditions.

Podocytes

[0269] Under static conditions the actin filaments of the podocytes were very long and followed a parallel direction. Under flow conditions, the actin filaments were a lot shorter and looked more like a star shape with a centre from which the filaments spread out.

Endothelial Cells

[0270] Actin filaments displayed a radial arrangement under flow conditions, but this was not as pronounced as in the podocytes. Under static conditions, PECAM1 appeared long and thin, and under flow conditions PECAM distribution was more like patches.

Podocytes and Endothelial Cells

[0271] Podocytes were very large under flow conditions, and were a lot smaller under static conditions. The cell types intermingled more under static conditions, which under flow conditions, cell types grouped together more (FIG. 4).

Example 5: Analysis of Podocytes Under Flow Conditions by Electron Microscopy

[0272] Podocytes having stable expression FLAG-tagged nephrin (passage 18+13) were cultured for 5 days at 37 C., under flow condition or under static conditions, and analysed by transmission electron microscopy (TEM). The cell surface was smoother following culture under static conditions than under flow conditions, and cells cultured under flow conditions had concentrations of actin fibres (FIG. 5 arrows).

Example 6: Printing of Cell Layers

[0273] Cell layers are printed to culture substrates by an inkjet printing method (e.g. see Saunders et al., Delivery of human fibroblast cells by piezoelectric drop-on-demand inkjet printing. Biomaterials 29 (2008) 193-203).

[0274] Endothelial cells are deposited in an array pattern on the culture substrate. Following culture of endothelial cells, podocytes are deposited on the endothelial cell layer. Cells are deposited in fixed patterns to form cell sheets of desired shape.

Example 7Production of Cell Sheets

Materials:

[0275] Conditionally immortalized glomerular endothelial cells (ciGEnC) [0276] Conditionally immortalized podocytes (glomerular visceral epithelial cells) [0277] Culture flasks (Corning 75 cm.sup.2 U-Shaped Canted Neck Cell Culture Flask with Vent Cap) [0278] Endothelial growth medium 2 (EGM2-MV, Cambrex, Wokingham, UK) [0279] RPMI 1640 medium (RPMI 1640 w/L-Glutamine-Bicarbonate, R8758, Sigma) [0280] Fetal bovine serum (heat inactivated, Gibco) [0281] Insulin-Transferrin-Selenium 100, Gibco, Ref. 41400-045 [0282] Temperature-responsive dishes (Nunc UpCell Surface cell culture dish, 3.5 cm culture dish, vented, VWR) [0283] PBS (Dulbecco's phosphate buffered saline, D8537, without calcium chloride and magnesium chloride, Sigma) [0284] Trypsin/EDTA solution (T3924, Sigma) [0285] Falcon tubes (Corning 50 mL PP Centrifuge Tubes, Conical Bottom with CentriStar Cap, Rack Packed, Sterile, Product #430828) [0286] Costar 6 Well Clear TC-Treated Multiple Well Plates, Individually Wrapped, Sterile, Product #3516) [0287] Coverslips (Coverglass, No 1 thickness, 13 mm diameter, PFM Medical UK ltd) [0288] ACLAR Embedding Film [0289] 4% Paraformaldehyde in phosphate buffered saline [0290] Transmission Electron microscopy (TEM) fixative: [0291] 4% Paraformaldehyde [0292] 2.5% Glutaraldehyde, [0293] 0.1M Hepes [0294] 70% Ethanol [0295] Sterile tweezers, pipette tips, stripettes

Method:

[0296] Procedure is performed under sterile conditions unless otherwise stated. [0297] 1. Culturing of conditionally immortalized glomerular endothelial cells (ciGEnC): [0298] Set up 1 culture flask of ciGEnCs (Corning 75 cm.sup.2 U-Shaped Canted Neck Cell Culture Flask with Vent Cap) for every 2 temperature-responsive dishes (Nunc UpCell Surface cell culture dish, 3.5 cm culture dish, vented, VWR). [0299] Cells were cultured in endothelial growth medium 2 (EGM2-MV, Cambrex, Wokingham, UK) containing fetal calf serum (5%) and growth factors as supplied, excepting VEGF. [0300] Grow cells at a temperature of 33 C. until they become fully confluent. Change endothelial growth medium every 2 to 3 days (10-15 ml medium per 75 cm.sup.2 culture flask). [0301] 2. Culturing of Podocytes: [0302] Set up 1 culture flask (Corning 75 cm.sup.2 U-Shaped Canted Neck Cell Culture Flask with Vent Cap) of podocytes in RPMI 1640 medium (RPMI 1640 w/L-Glutamine-Bicarbonate, R8758, Sigma) supplemented with 10% fetal bovine serum (heat inactivated, Gibco) and 1 Insulin-Transferrin-Selenium (Insulin-Transferrin-Selenium 100, Gibco, Ref. 41400-045). [0303] Grow cells at a temperature of 33 C. and change medium every 2 to 3 days. [0304] 3. Seeding conditionally immortalized glomerular endothelial cells onto temperature-responsive dishes: [0305] When ciGEnC reach high confluency (app. 3 000 000 cells/flask), rinse cells 2 with 10 ml of phosphate buffered saline (Dulbecco's phosphate buffered saline, D8537, without calcium chloride and magnesium chloride, Sigma). [0306] Add 3-5 ml of Trypsin/EDTA solution (T3924, Sigma) and incubate at 37 C. for 5 minutes to detach the cells. Tap flask to help with detachment, add 5-7 ml of supplemented endothelial growth medium 2 to the flask to inactivate the Trypsin. [0307] Transfer cell suspension to a falcon tube and spin for 4 minutes at 1600 rpm. [0308] Remove supernatant and resuspend cell pellet in 6 ml of fresh cell culture media. Add 3 ml of the cell suspension to each temperature-responsive dish and incubate at 33 C. until whole layer of cells has formed. [0309] Change media every 2-3 days. Usually it takes about a week for the cells to become confluent enough to yield a cell sheet. [0310] 4. Seeding conditionally immortalized podocytes onto coverslips and ACLAR Film: [0311] Split one confluent 75 cm.sup.2 culture flask with conditionally immortalized podocytes (approx. 2.5 million cells/flask) onto glass coverslips and ACLAR Film in a 6-well cell culture plate (on the same day as you seed the ciGEnC onto temperature-responsive dishes). [0312] Rinse cells once with 10 ml of phosphate buffered saline (Dulbecco's phosphate buffered saline, D8537, without calcium chloride and magnesium chloride, Sigma). [0313] Add 3-5 ml of Trypsin/EDTA solution (T3924, Sigma) and incubate at 37 C. for 5 minutes to detach the cells. Tap flask to help with detachment, add 5-7 ml supplemented RPMI 2 medium to the flask to inactivate the Trypsin. [0314] Transfer cell suspension to a falcon tube and spin for 4 minutes at 1600 rpm. [0315] Remove supernatant and resuspend cell pellet in 18 ml of fresh cell culture media. [0316] Prepare 6 well culture dish: [0317] Cut out 3 squares from the ACLAR Film (need to fit inside well of the culture plate) [0318] Sterilize in 70% Ethanol [0319] Sterilize 3 glass cover slips in 70% Ethanol [0320] Rinse in phosphate buffered saline [0321] Add each square/coverslip into one well of the culture plate [0322] Add 3 ml of podocyte cell suspension to each well of the culture plate. [0323] Incubate at 33 C., change RPMI 1640 medium every 2-3 days. [0324] 5. Harvesting cell sheets from temperature-responsive dishes: [0325] When ciGEnCs on temperature-responsive dish reach high confluency state, move dish to room temperature, remove medium and add PVDF membrane (part of the kit) with sterile tweezers (sterilize with 70% Ethanol before use). [0326] Add couple of drops of cold medium (cells don't try out and PVDF doesn't float off), leave for a minimum of 30 min at room temperature [0327] Loosen edges with sterile tweezers and pull cell sheet towards PVDF membrane, check under microscope to see if cell sheet has loosened (leave longer if not). Grab PVDF with tweezers and lift up both, cell sheet and membrane from dish. Check dish under microscope if cells are gone. [0328] Remove medium from podocytes grown on cover slips and ACLAR Film inside a 6-well culture plate. Podocytes reach very high confluency at that point. [0329] Cut cell sheet and membrane with scissors in two halves (cell sheets can be left in one piece if preferred). Put each half onto the podocytes. Add small amount of endothelial medium onto cell sheet to avoid cells drying out. [0330] Incubate at 33 C. for 30 min to let cell sheet attach to podocytes, add more endothelial medium and let PVDF membrane come off on its own. Incubate dishes at 37 C. for 14 days. Change media regularly every 2 to 3 days. [0331] 6. Fixation of cells: [0332] Fix glass cover slips with 4% Paraformaldehyde solution for 15 min. Remove fixative and add PBS. Store in fridge. Samples are ready for Immunocytochemistry staining. [0333] ACLAR: Don't remove medium, add as much TEM fixative as medium to wells and leave in fix, never let ACLAR dry out. Store in fridge until samples can be processed for Transmission Electron Microscopy.