CARRIER FOR CELL CULTURING AND A METHOD OF PREPARATION THEREOF

Abstract

A carrier for cell culturing that contains a multiwell plate, where at least one of the wells of the multiwell plate has a porous substrate having the porosity of 90% and adapted for cell culturing, and the porous substrate adheres to the surface of the well. A method for preparation of the carrier for cell culturing is also provided.

Claims

1. A carrier for cell culturing, which contains a multiwell plate, wherein at least one of the wells of the said multiwell plate contains a porous substrate, preferably having the porosity of 90% and adapted for cell culturing, and wherein the said porous substrate adheres to the surface of the said well.

2. The carrier according to claim 1, wherein the porous substrate is provided in at least 25% of the wells, more preferably in at least 50% of the wells or in all the wells of the multiwell plate.

3. The carrier according to claim 1, wherein the multiwell plate is made of a material selected from the group consisting of plastics, glass, ceramics, metals, and combinations of these materials.

4. The carrier according to claim 1, wherein the porous substrate is composed of at least two layers which differ mutually in at least one of: material composition, presence or absence of additives, composition of additives, porosity, pore size, pore connectivity.

5. The carrier according to claim 1, wherein the pore size of the porous substrate is within the range from 0.1 to 1000 m, preferably between 5 and 1000 m or between 100 and 2000 nm or within the range from 50 to 600 m.

6. The carrier according to claim 1, wherein the material of the porous substrate is selected from the group consisting of proteins of the extracellular matrix (ECM); structural proteins such as collagens, fibrin, silk fibroin, elastin or gelatine; polysaccharides such as hyaluronic acid and its derivatives, chitosan and its derivatives, starches, resinous gums, cellulose and its derivatives; resins; biocompatible synthetic polymers, such as polylactic acid, polyglycolic acid, polyethyleneglycol and their block copolymers, polycaprolaktone, polyhydroxybutyrate, polyurethanes; natural inorganic nanotubes; and combinations of these materials; wherein the porous substrate material optionally is cross-linked, and/or the porous substrate material optionally contains as additives: bioactive proteins, their fragments or mixtures, phosphates, organic or inorganic nano-particles such as hydroxyapatite, alpha- or beta-tricalcium phosphate, oxide ceramics, such as SiO.sub.2, ZnO, metal nanoparticles such as Ag, Au, Mg, or metal nano-particles decorated with covalently or physically bound organic ligands.

7. The carrier according to claim 1, wherein the porous substrate has the smallest dimension of at least 10 micrometers, preferably at least 100 micrometers, more preferably at least 0.5 mm, most preferably at least 1 to 10 mm.

8. A method for preparation of a carrier for cell culturing, the said method comprising the following sequence of steps: applying a layer of solvent into at least one well of the multiwell plate and freezing the layer of solvent, applying at least one layer of a material for a porous substrate into the said at least one well of the multiwell plate, and freezing each layer after it is applied, applying a cover layer of the solvent into the said at least one well of the multiwell plate, followed by freezing the cover layer, and freeze drying after applying each layer separately, or after applying all the layers into the said at least one well.

9. The method according to claim 8, wherein the the freezing is performed in two steps: first freezing to a temperature between 5 and 40 C., followed by freezing to a temperature between 20 and 60 C.

10. The method according to claim 8, wherein freeze drying is carried out at a temperature lower than 70 C., preferably at lower than atmospheric pressure, more preferably at a pressure of 1000 Pa or less.

11. The method according to claim 8, wherein the material of the porous substrate is applied in the form of a solution, suspension, aerogel, emulsion, hydrogel, microparticles or nano-fibers.

12. The method according to claim 8, wherein the material of the porous substrate is cross-linked using a cross-linking agent.

13. A method of culturing cells, comprising the steps of: providing a carrier according to claim 1, seeding cells on the porous substrates in the said carrier, culturing cells on the the porous substrates of the said carrier, optionally subjecting the cultured cells to a chemical, physical or biological analysis.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0039] FIG. 1 shows SEM micrographs of the surface of the surface of the porous 3D collagenous substrate. Left according to the example 1 (0.5 wt. % of collagen I) and right according to example 2 (combination of 0.5 a 2 wt. % collagen I layers).

[0040] FIG. 2 shows the composition dependence of the pore size of the 3D collagen I substrate (left), Effect of the HAP concentration at constant collagen I content (right).

[0041] FIG. 3 shows nanoCT scan of the porous substrate according to example 3 (grey area) and its 3D colonization by cells of lung cancer (dark grey circular objects).

[0042] FIG. 4 shows fluorescence confocal microscopy of differentiated MSC cells on the porous substrate from example 1. Survival rate of the cells is documented by staining with BCECF-AM/propidiumjodide 28 days after seeding the porous substrate containing 0.5 wt. % collagen I and (a) 50 wt. % nHAP, (b) 70 wt. % nHAP and (c) containing 2 wt. % collagen I and 50 wt. % of nHAP Immunofluorescence detection of osteocalcin 28 days after seeding the same substrates.

[0043] FIG. 5 shows SEM micrographs of the 3D substrate from example 6 (left) containing hyaluronic acid according and containing chitosan according to example 7.

[0044] FIG. 6 shows fluorescence confocal microscopy of differentiated cells on the 3D substrate from the example 8 (left) and 9 (right). Survival rate of cells is documented by staining their cytoplasm with green BCECF-AM/propidiumiodide and their nuclei in red with propidium iodide.

[0045] FIG. 7 shows fluorescence confocal microscopy of differentiated cells on the porous 3D substrate from example 10 (left) and 11 (right). Survival rate of cells is documented by staining their cytoplasm with green BCECF-AM/propidiumiodide and their nuclei in red with propidium iodide.

EXAMPLES OF CARRYING OUT THE INVENTION

Example 1

[0046] Wells in a 48-well plate were layer by layer filled with a biocompatible material in the following sequence: [0047] 1. Ultrapure water (Milipore, purity II) [0048] 2. Layers of homogeneous solutions of bovine collagen I, concentration ranging from 0.3 to 3 wt. % in ultrapure water [0049] 3. Ultrapure water (Milipore, purity II).

[0050] The materials in the wellplate were frozen under controlled conditions after application of each layer first at 18 C. (1 hour), then at 35 C. (1 hour), and, finally, freeze dried at the condensator temperature of 95 C. and pressure lower than 1000 Pa to constant weight. Following freeze drying, collagen is cross-linked using EDC/NHS system directly in the well and, again, freeze dried under the same conditions as described above. The wells contain homogeneous 3D porous substrate with pore size engineered to the range of 250-450 m and thickness of 5 mm adhering to the well walls. Even after swelling, the substrate remains attached to the well bottom without detaching and floating (see FIG. 1). Porosity in the layers was proportional to the collagen concentration and was within the interval from 99.5% for the 0.3 wt. % collagen I concentration to 97% for collagen concentration of 3 wt. %. Porosity was determined by image analysis of the 3D morphology of the freeze dried substrate obtained employing the microCT computer tomograph (for example Nano3DX Rigaku, Japan). The porosities shown above are the average values calculated from 5 repetitive measurements performed for randomly selected wells from the wellplate. Standard deviation of the measurements was less than 10% of the average value. This substrate is, thus, suitable for cell seeding and culturing and has already been subject to in-vitro and in-vivo tests with various cell types.

Example 2

[0051] Wells in a 48-well plate were layer by layer filled with a biocompatible material in the following sequence: [0052] 1. Ultrapure water (Milipore, purity II) [0053] 2. Layer of homogeneous solutions of bovine collagen I, concentration of 2 wt. % in ultrapure water [0054] 3. Layer of homogeneous solutions of bovine collagen I, concentration of 0.5 wt. % in ultrapure water [0055] 4. Ultrapure water (Milipore, purity II).

[0056] The materials in the wellplate were frozen under controlled conditions after deposition of each layer first at 18 C. (1 hour), then at 35 C. (1 hour), and, finally, freeze dried at the condensator temperature of 95 C. and pressure lower than 1000 Pa to constant weight. Following freeze drying, collagen is cross-linked using EDC/NHS system directly in the well and, again, freeze dried under the same conditions as described above. The wells contain homogeneous 3D porous substrate with the total thickness of 6 mm exhibiting controlled pore size of 100 m in the bottom layer 3 mm thick as well as in the top layer 3 mm thick exhibiting pore size of 250-450 m and adhering to the well walls. Even after swelling, the substrate remains attached to the well bottom without detaching and floating. This substrate is, thus, suitable for seeding and culturing broad range of cell types and has already been subject to in-vitro and in-vivo tests (see FIG. 2). Porosity in the bottom layer was equal to 98.5% for the 2 wt. % collagen I concentration and 99.3% for collagen concentration of 0.5 wt. %. Porosity was determined by image analysis of the 3D morphology of the freeze dried substrate obtained employing the microCT computer tomograph (for example Nano3DX Rigaku, Japan). The porosities shown above are the average values calculated from 5 repetitive measurements performed for randomly selected wells from the wellplate. Standard deviation of the measurements was less than 10% of the average value.

Example 3

[0057] Wells in a 96-well plate were layer by layer filled with a biocompatible material in the following sequence: [0058] 1. Ultrapure water (Milipore, purity II) [0059] 2. Layer of electrospun nanofibers consisting of the mixture of polycaprolactone and gelatine and having fiber diameter of 120-280 nm [0060] 3. Layer of homogeneous solutions of bovine collagen I, concentration of 0.5 wt. % in ultrapure water [0061] 4. Ultrapure water (Milipore, purity II).

[0062] The materials in the wellplate were frozen under controlled conditions after deposition of each layer first at 18 C. (1 hour), then at 35 C. (1 hour), and, finally, freeze dried at the condensator temperature of 95 C. and pressure lower than 1000 Pa to constant weight. Following freeze drying, collagen is cross-linked using EDC/NHS system directly in the well and, again, freeze dried under the same conditions as described above. The wells contain homogeneous 3D porous substrate with the total thickness of 3 mm exhibiting controlled pore size of 50 m in the bottom layer 0.5 mm thick as well as in the top layer 2.5 mm thick exhibiting pore size of 400 m and adhering to the well walls. Even after swelling, the substrate remains attached to the well bottom without detaching and floating. This substrate is, thus, suitable for seeding and culturing broad range of cell types and has already been subject to in-vitro and in-vivo tests. Growth of the cells throughout the entire volume of the 3D substrate is depicted in FIG. 3. Porosity in the bottom layer was equal to 45.3% for nanofibrous layer and 99.3% for collagen concentration of 0.5 wt. %. Porosity was determined by image analysis of the 3D morphology of the freeze dried substrate obtained employing the microCT computer tomograph (for example Nano3DX Rigaku, Japan). The porosities shown above are the average values calculated from 5 repetitive measurements performed for randomly selected wells from the wellplate. Standard deviation of the measurements was less than 10% of the average value.

Example 4

[0063] Wells in a 48-well plate were layer by layer filled with a biocompatible material in the following sequence: [0064] 1. Ultrapure water (Milipore, purity II) [0065] 2. Layer of homogeneous solutions of bovine collagen I, concentration of 0.5 wt. % in ultrapure water [0066] 3. Layer of homogeneous solution of bovine collagen I with collagen concentration of 0.5 wt. % and 50 or 70 wt. % per dry collagen weight of nano hydroxyapatite (nHAP) in ultrapure water [0067] 4. Ultrapure water (Milipore, purity II).

[0068] The materials in the wellplate were frozen under controlled conditions after deposition of each layer first at 18 C. (1 hour), then at 35 C. (1 hour), and, finally, freeze dried at the condensator temperature of 95 C. and pressure lower than 1000 Pa to constant weight. Following freeze drying, collagen is cross-linked using EDC/NHS system directly in the well and, again, freeze dried under the same conditions as described above. The wells contain homogeneous 3D porous substrate with the total thickness of 5 mm exhibiting controlled pore size of 400 m in the bottom layer 2 mm thick as well as in the top layer 3 mm thick exhibiting pore size of 350 m and adhering to the well walls. Even after swelling, the substrate remains attached to the well bottom without detaching and floating. This substrate is, thus, suitable for seeding and culturing broad range of cell types and has already been subject to in-vitro and in-vivo tests (FIG. 4-a,b,e,d). Porosity in the bottom layer was equal to 99.3% and was ranging between 97 and 98% for the top collagen/nHAP layer depending on the nHAP content. Porosity was determined by image analysis of the 3D morphology of the freeze dried substrate obtained employing the microCT computer tomograph (for example Nano3DX Rigaku, Japan). The porosities shown above are the average values calculated from 5 repetitive measurements performed for randomly selected wells from the wellplate. Standard deviation of the measurements was less than 10% of the average value.

Example 5

[0069] Wells in a 96-well plate were layer by layer filled with a biocompatible material in the following sequence: [0070] 1. Ultrapure water (Milipore, purity II) [0071] 2. Layer of homogeneous solutions of bovine collagen I, concentration of 2 wt. % in ultrapure water [0072] 3. Layer of homogeneous solution of bovine collagen I with collagen concentration of 0.5 wt. % and 50 wt. % per dry collagen weight of nano hydroxyapatite (nHAP) in ultrapure water [0073] 4. Ultrapure water (Milipore, purity II).

[0074] The materials in the wellplate were frozen under controlled conditions after deposition of each layer first at 18 C. (1 hour), then at 35 C. (1 hour), and, finally, freeze dried at the condensator temperature of 95 C. and pressure lower than 1000 Pa to constant weight. Following freeze drying, collagen is cross-linked using EDC/NHS system directly in the well and, again, freeze dried under the same conditions as described above. The wells contain homogeneous 3D porous substrate with the total thickness of 4 mm exhibiting controlled pore size of 100 m in the bottom layer 1 mm thick as well as in the top layer 3 mm thick exhibiting pore size of 350 m and adhering to the well walls. Even after swelling, the substrate remains attached to the well bottom without detaching and floating. This substrate is, thus, suitable for seeding and culturing broad range of cell types and has already been subject to in-vitro and in-vivo tests (FIG. 4-c,f). Porosity in the bottom layer was equal to 98.5% and was 98% for the top collagen/nHAP layer. Porosity was determined by image analysis of the 3D morphology of the freeze dried substrate obtained employing the microCT computer tomograph (for example Nano3DX Rigaku, Japan). The porosities shown above are the average values calculated from 5 repetitive measurements performed for randomly selected wells from the wellplate. Standard deviation of the measurements was less than 10% of the average value.

Example 6

[0075] Wells in a 96-well plate were layer by layer filled with a biocompatible material in the following sequence: [0076] 1. Ultrapure water (Milipore, purity II) [0077] 2. Layer of homogeneous solutions of bovine collagen I, concentration of 2 wt. % in ultrapure water [0078] 3. Layer of homogeneous solution of bovine collagen I with collagen concentration of 0.5 wt. % and 10 wt. % per dry collagen weight of hyaluronic acid (HA) in ultrapure water [0079] 4. Ultrapure water (Milipore, purity II).

[0080] The materials in the wellplate were frozen under controlled conditions after deposition of each layer first at 18 C. (1 hour), then at 35 C. (1 hour), and, finally, freeze dried at the condensator temperature of 95 C. and pressure lower than 1000 Pa to constant weight. Following freeze drying, collagen is cross-linked using EDC/NHS system directly in the well and, again, freeze dried under the same conditions as described above. The wells contain homogeneous 3D porous substrate with the total thickness of 4 mm exhibiting controlled pore size of 400 m in the bottom layer 3 mm thick as well as in the top layer 1 mm thick exhibiting pore size of 520 m and adhering to the well walls. Even after swelling, the substrate remains attached to the well bottom without detaching and floating. This substrate is, thus, suitable for seeding and culturing broad range of cell types and has already been subject to in-vitro and in-vivo tests (FIG. 5a). Porosity in the bottom layer was equal to 99.5% and was 98.5% for the top collagen/HA layer. Porosity was determined by image analysis of the 3D morphology of the freeze dried substrate obtained employing the microCT computer tomograph (for example Nano3DX Rigaku, Japan). The porosities shown above are the average values calculated from 5 repetitive measurements performed for randomly selected wells from the wellplate. Standard deviation of the measurements was less than 10% of the average value.

Example 7

[0081] Wells in a 48-well plate were layer by layer filled with a biocompatible material in the following sequence: [0082] 1. Ultrapure water (Milipore, purity II) [0083] 2. Layer of homogeneous solutions of bovine collagen I, concentration of 2 wt. % in ultrapure water [0084] 3. Layer of homogeneous solution of bovine collagen I with collagen concentration of 0.5 wt. % and 20 wt. % per dry collagen weight of chitosan in ultrapure water [0085] 4. Ultrapure water (Milipore, purity II).

[0086] The materials in the wellplate were frozen under controlled conditions after deposition of each layer first at 18 C. (1 hour), then at 35 C. (1 hour), and, finally, freeze dried at the condensator temperature of 95 C. and pressure lower than 1000 Pa to constant weight. Following freeze drying, collagen is cross-linked using EDC/NHS system directly in the well and, again, freeze dried under the same conditions as described above. The wells contain homogeneous 3D porous substrate with the total thickness of 8 mm exhibiting controlled pore size of 100 m in the bottom layer 2 mm thick as well as in the top layer 6 mm thick exhibiting pore size of 600 m and adhering to the well walls. Even after swelling, the substrate remains attached to the well bottom without detaching and floating. This substrate is, thus, suitable for seeding and culturing broad range of cell types and has already been subject to in-vitro and in-vivo tests (FIG. 5b). Porosity in the bottom layer was equal to 98.5% and was 98% for the top collagen/chitosan layer. Porosity was determined by image analysis of the 3D morphology of the freeze dried substrate obtained employing the microCT computer tomograph (for example Nano3DX Rigaku, Japan). The porosities shown above are the average values calculated from 5 repetitive measurements performed for randomly selected wells from the wellplate. Standard deviation of the measurements was less than 10% of the average value.

Example 8

[0087] Wells in a 24-well plate were layer by layer filled with a biocompatible material in the following sequence: [0088] 1. Ultrapure water (Milipore, purity II) [0089] 2. Layer of homogeneous solutions of bovine collagen I, concentration of 0.5 wt. % in ultrapure water [0090] 3. Layer of homogeneous solution of bovine collagen I with collagen concentration of 0.5 wt. % and 30 wt. % per dry collagen weight of oxidized cellulose in ultrapure water [0091] 4. Ultrapure water (Milipore, purity II).

[0092] The materials in the wellplate were frozen under controlled conditions after deposition of each layer first at 18 C. (1 hour), then at 35 C. (1 hour), and, finally, freeze dried at the condensator temperature of 95 C. and pressure lower than 1000 Pa to constant weight. Following freeze drying, collagen is cross-linked using EDC/NHS system directly in the well and, again, freeze dried under the same conditions as described above. The wells contain homogeneous 3D porous substrate with the total thickness of 10 mm exhibiting controlled pore size of 400 m in the bottom layer 4 mm thick as well as in the top layer 6 mm thick exhibiting pore size of 350 m and adhering to the well walls. Even after swelling, the substrate remains attached to the well bottom without detaching and floating. This substrate is, thus, suitable for seeding and culturing broad range of cell types and has already been subject to in-vitro and in-vivo tests (FIG. 6a). Porosity in the bottom layer was equal to 99.5% and was 98.3% for the top collagen/chitosan layer. Porosity was determined by image analysis of the 3D morphology of the freeze dried substrate obtained employing the microCT computer tomograph (for example Nano3DX Rigaku, Japan). The porosities shown above are the average values calculated from 5 repetitive measurements performed for randomly selected wells from the wellplate. Standard deviation of the measurements was less than 10% of the average value.

Example 9

[0093] Wells in a 96-well plate were layer by layer filled with a biocompatible material in the following sequence: [0094] 1. Ultrapure water (Milipore, purity II) [0095] 2. Layer of homogeneous solutions of bovine collagen I, concentration of 2 wt. % in ultrapure water [0096] 3. Layer of homogeneous solution of bovine collagen I with collagen concentration of 0.5 wt. % and 15 wt. % per dry collagen weight of blood platelet lysate in ultrapure water [0097] 4. Ultrapure water (Milipore, purity II).

[0098] The materials in the wellplate were frozen under controlled conditions after deposition of each layer first at 18 C. (1 hour), then at 35 C. (1 hour), and, finally, freeze dried at the condensator temperature of 95 C. and pressure lower than 1000 Pa to constant weight. Following freeze drying, collagen is cross-linked using EDC/NHS system directly in the well and, again, freeze dried under the same conditions as described above. The wells contain homogeneous 3D porous substrate with the total thickness of 6 mm exhibiting controlled pore size of 400 m in the bottom layer 3 mm thick as well as in the top layer 3 mm thick exhibiting pore size of 420 m and adhering to the well walls. Even after swelling, the substrate remains attached to the well bottom without detaching and floating. This substrate is, thus, suitable for seeding and culturing broad range of cell types and has already been subject to in-vitro and in-vivo tests (FIG. 6b). Porosity in the bottom layer was equal to 99.5% and was 99% for the top collagen/chitosan layer. Porosity was determined by image analysis of the 3D morphology of the freeze dried substrate obtained employing the microCT computer tomograph (for example Nano3DX Rigaku, Japan). The porosities shown above are the average values calculated from 5 repetitive measurements performed for randomly selected wells from the wellplate. Standard deviation of the measurements was less than 10% of the average value.

Example 10

[0099] Wells in a 24-well plate were layer by layer filled with a biocompatible material in the following sequence: [0100] 1. Ultrapure water (Milipore, purity II) [0101] 2. Layer of homogeneous solutions of bovine collagen I, concentration of 0.5 wt. % in ultrapure water [0102] 3. Layer of homogeneous solution of bovine collagen I with collagen concentration of 0.5 wt. % and 20 wt. % per dry collagen weight of chitin and 10 wt. % per dry collagen weight of blood platelet lysate in ultrapure water [0103] 4. Ultrapure water (Milipore, purity II).

[0104] The materials in the wellplate were frozen under controlled conditions after deposition of each layer first at 18 C. (1 hour), then at 35 C. (1 hour), and, finally, freeze dried at the condensator temperature of 95 C. and pressure lower than 1000 Pa to constant weight. Following freeze drying, collagen is cross-linked using EDC/NHS system directly in the well and, again, freeze dried under the same conditions as described above. The wells contain homogeneous 3D porous substrate with the total thickness of 10 mm exhibiting controlled pore size of 400 m in the bottom layer 3 mm thick as well as in the top layer 7 mm thick exhibiting pore size of 380 m and adhering to the well walls. Even after swelling, the substrate remains attached to the well bottom without detaching and floating. This substrate is, thus, suitable for seeding and culturing broad range of cell types and has already been subject to in-vitro and in-vivo tests (FIG. 6b). Porosity in the bottom layer was equal to 99.5% and was 98.5% for the top collagen/chitosan/BPL layer. Porosity was determined by image analysis of the 3D morphology of the freeze dried substrate obtained employing the microCT computer tomograph (for example Nano3DX Rigaku, Japan). The porosities shown above are the average values calculated from 5 repetitive measurements performed for randomly selected wells from the wellplate. Standard deviation of the measurements was less than 10% of the average value.

Example 11

[0105] Wells in a 96-well plate were layer by layer filled with a biocompatible material in the following sequence: [0106] 1. Ultrapure water (Milipore, purity II) [0107] 2. Layer of homogeneous solutions of bovine collagen I, concentration of 2 wt. % in ultrapure water [0108] 3. Layer of homogeneous solution of bovine collagen I with collagen concentration of 0.5 wt. % and 20 wt. % per dry collagen weight of oxidized cellulose and 10 wt. % per dry weight of collagen of blood platelet lysate in ultrapure water [0109] 4. Ultrapure water (Milipore, purity II).

[0110] The materials in the wellplate were frozen under controlled conditions after deposition of each layer first at 18 C. (1 hour), then at 35 C. (1 hour), and, finally, freeze dried at the condensator temperature of 95 C. and pressure lower than 1000 Pa to constant weight. Following freeze drying, collagen is cross-linked using EDC/NHS system directly in the well and, again, freeze dried under the same conditions as described above. The wells contain homogeneous 3D porous substrate with the total thickness of 6 mm exhibiting controlled pore size of 400 m in the bottom layer 2 mm thick as well as in the top layer 4 mm thick exhibiting pore size of 250 m and adhering to the well walls. Even after swelling, the substrate remains attached to the well bottom without detaching and floating. This substrate is, thus, suitable for seeding and culturing broad range of cell types and has already been subject to in-vitro and in-vivo tests (FIG. 6b). Porosity in the bottom layer was equal to 99.5% and was 98% for the top collagen/OC/BPL layer. Porosity was determined by image analysis of the 3D morphology of the freeze dried substrate obtained employing the microCT computer tomograph (for example Nano3DX Rigaku, Japan). The porosities shown above are the average values calculated from 5 repetitive measurements performed for randomly selected wells from the wellplate (FIG. 7). Standard deviation of the measurements was less than 10% of the average value.

Example 12

[0111] Wells in a 24-well plate were layer by layer filled with a biocompatible material in the following sequence: [0112] 1. Ultrapure water (Milipore, purity II) [0113] 2. Layer of homogeneous solutions of decellularized collagen tissue, concentration of 0.7 wt. % containing remnants of bioactive proteins in ultrapure water [0114] 3. Layer of homogeneous solutions of decellularized collagen tissue, concentration of 0.5 wt. % containing remnants of bioactive proteins in ultrapure water [0115] 4. Ultrapure water (Milipore, purity II).

[0116] The materials in the wellplate were frozen under controlled conditions after deposition of each layer first at 18 C. (1 hour), then at 35 C. (1 hour), and, finally, freeze dried at the condensator temperature of 95 C. and pressure lower than 1000 Pa to constant weight. The wells contain homogeneous 3D porous substrate with the total thickness of 10 mm exhibiting controlled pore size of 300 m in the bottom layer 2 mm thick as well as in the top layer 8 mm thick exhibiting pore size of 400 m and adhering to the well walls. Even after swelling, the substrate remains attached to the well bottom without detaching and floating. This substrate is, thus, suitable for seeding and culturing broad range of cell types and has already been subject to in-vitro and in-vivo tests (FIG. 6b). Porosity in the bottom layer was equal to 88.5% and was 91% for the top layer. Porosity was determined by image analysis of the 3D morphology of the freeze dried substrate obtained employing the microCT computer tomograph (for example Nano3DX Rigaku, Japan). The porosities shown above are the average values calculated from 5 repetitive measurements performed for randomly selected wells from the wellplate. Standard deviation of the measurements was less than 10% of the average value.

Example 13

[0117] Wells in a 24-well plate were layer by layer filled with a biocompatible material in the following sequence: [0118] 1. Ultrapure water (Milipore, purity II) [0119] 2. Layer of homogeneous solutions of decellularized collagen tissue, concentration of 0.7 wt. % containing remnants of bioactive proteins in ultrapure water [0120] 3. Layer of homogeneous solutions of decellularized collagen tissue, concentration of 0.5 wt. % containing remnants of bioactive proteins and enriched with 2 g TGF- growth factor in ultrapure water [0121] 4. Ultrapure water (Milipore, purity II).

[0122] The materials in the wellplate were frozen under controlled conditions after deposition of each layer first at 18 C. (1 hour), then at 35 C. (1 hour), and, finally, freeze dried at the condensator temperature of 95 C. and pressure lower than 1000 Pa to constant weight. The wells contain homogeneous 3D porous substrate with the total thickness of 10 mm exhibiting controlled pore size of 300 m in the bottom layer 5 mm thick as well as in the top layer 5 mm thick exhibiting pore size of 400 m and adhering to the well walls. Even after swelling, the scaffold remains attached to the well bottom without detaching and floating. This substrate is, thus, suitable for seeding and culturing broad range of cell types and has already been subject to in-vitro and in-vivo tests. Porosity in the bottom layer was equal to 88.5% and was 91% for the top layer. Porosity was determined by image analysis of the 3D morphology of the freeze dried substrate obtained employing the microCT computer tomograph (for example Nano3DX Rigaku, Japan). The porosities shown above are the average values calculated from 5 repetitive measurements performed for randomly selected wells from the wellplate. Standard deviation of the measurements was less than 10% of the average value.

Example 14

[0123] Wells in a 24-well plate were layer by layer filled with a biocompatible material in the following sequence: [0124] 1. Ultrapure water (Milipore, purity II) [0125] 2. Layer of homogeneous solutions of collagen I, concentration of 0.5 wt. % in ultrapure water [0126] 3. Layer of randomly oriented nanofibers from halloysite modified gelatine 180 nm in diameter with 20 wt. % areal concentration in ultrapure water [0127] 4. Ultrapure water (Milipore, purity II).

[0128] The materials in the wellplate were frozen under controlled conditions after deposition of each layer first at 18 C. (1 hour), then at 35 C. (1 hour), and, finally, freeze dried at the condensator temperature of 95 C. and pressure lower than 1000 Pa to constant weight. Following freeze drying, collagen is cross-linked using EDC/NHS system directly in the well and, again, freeze dried under the same conditions as described above. The wells contain homogeneous 3D porous substrate with the total thickness of 4 mm exhibiting controlled pore size of 400 m in the bottom layer 2 mm thick as well as in the top layer 2 mm thick exhibiting pore size of 50 m and adhering to the well walls. Even after swelling, the substrate remains attached to the well bottom without detaching and floating. This substrate is, thus, suitable for seeding and culturing broad range of cell types and has already been subject to in-vitro and in-vivo tests. Porosity in the bottom layer was equal to 99.3% and was 75% for the top layer. Porosity was determined by image analysis of the 3D morphology of the freeze dried substrate obtained employing the microCT computer tomograph (for example Nano3DX Rigaku, Japan). The porosities shown above are the average values calculated from 5 repetitive measurements performed for randomly selected wells from the wellplate. Standard deviation of the measurements was less than 10% of the average value.

Example 15

[0129] Wells in a 48-well plate were layer by layer filled with a biocompatible material in the following sequence: [0130] 1. Ultrapure water (Milipore, purity II) [0131] 2. Layer of micellar gel from PLA-PGA-PEG block copolymer, concentration of 1 wt. % in ultrapure water [0132] 3. Layer of homogeneous chitosan solution, concentration of 2 wt. % in ultrapure water [0133] 4. Ultrapure water (Milipore, purity II).

[0134] The materials in the wellplate were frozen under controlled conditions after deposition of each layer first at 18 C. (1 hour), then at 35 C. (1 hour), and, finally, freeze dried at the condensator temperature of 95 C. and pressure lower than 1000 Pa to constant weight. The wells contain homogeneous 3D porous substrate with the total thickness of 4 mm exhibiting controlled pore size of 400 m in the bottom layer 2 mm thick as well as in the top layer 2 mm thick exhibiting pore size of 380 m and adhering to the well walls. Even after swelling, the substrate remains attached to the well bottom without detaching and floating. This substrate is, thus, suitable for seeding and culturing broad range of cell types and has already been subject to in-vitro and in-vivo tests. Porosity in the bottom layer was equal to 98.2% and was 98.8% for the top layer. Porosity was determined by image analysis of the 3D morphology of the freeze dried substrate obtained employing the microCT computer tomograph (for example Nano3DX Rigaku, Japan). The porosities shown above are the average values calculated from 5 repetitive measurements performed for randomly selected wells from the wellplate. Standard deviation of the measurements was less than 10% of the average value.

Example 16

[0135] Wells in a 24-well plate were layer by layer filled with a biocompatible material in the following sequence: [0136] 1. Ultrapure water (Milipore, purity II) [0137] 2. Layer of homogeneous solutions of decellularized collagen tissue, concentration of 0.7 wt. % containing remnants of bioactive proteins in ultrapure water [0138] 3. Layer of randomly oriented nanofibers from halloysite modified gelatine 180 nm in diameter with 20 wt. % areal concentration in ultrapure water [0139] 4. Layer of homogeneous solutions of collagen I, concentration of 0.5 wt. % in ultrapure water

[0140] 5. Ultrapure water (Milipore, purity II).

[0141] The materials in the wellplate were frozen under controlled conditions after deposition of each layer first at 18 C. (1 hour), then at 35 C. (1 hour), and, finally, freeze dried at the condensator temperature of 95 C. and pressure lower than 1000 Pa to constant weight. Following freeze drying, collagen is cross-linked using EDC/NHS system directly in the well and, again, freeze dried under the same conditions as described above. The wells contain homogeneous 3D porous substrate with the total thickness of 4.5 mm exhibiting controlled pore size of 400 m in the bottom layer 2 mm thick as well as in the top layer 2 mm thick exhibiting pore size of 380 m and adhering to the well walls. The middle layer 0.5 mm thick separating the top and bottom layers exhibited pore size of 80 m. Even after swelling, the substrate remains attached to the well bottom without detaching and floating. This substrate is, thus, suitable for seeding and culturing broad range of cell types and has already been subject to in-vitro and in-vivo tests. Porosity in the bottom layer was equal to 90.3%, 99.3% for the top layer and 75% for the nanofibrous layer. Porosity was determined by image analysis of the 3D morphology of the freeze dried substrate obtained employing the microCT computer tomograph (for example Nano3DX Rigaku, Japan). The porosities shown above are the average values calculated from 5 repetitive measurements performed for randomly selected wells from the wellplate. Standard deviation of the measurements was less than 10% of the average value.

Example 17

[0142] Wells in a 96-well plate were layer by layer filled with a biocompatible material in the following sequence: [0143] 1. Ultrapure water (Milipore, purity II) [0144] 2. Layer of homogeneous collagen solution, concentration of 2 wt. % in ultrapure water [0145] 3. Layer of of 2 wt. % gum-karraya modified with 20 wt. % of oxidized cellulose per weight of gum-karaya in ultrapure water [0146] 4. Ultrapure water (Milipore, purity II).

[0147] The materials in the wellplate were frozen under controlled conditions after deposition of each layer first at 18 C. (1 hour), then at 35 C. (1 hour), and, finally, freeze dried at the condensator temperature of 95 C. and pressure lower than 1000 Pa to constant weight. Following freeze drying, collagen is cross-linked using EDC/NHS system directly in the well and, again, freeze dried under the same conditions as described above. The wells contain homogeneous 3D porous substrate with the total thickness of 4 mm exhibiting controlled pore size of 400 m in the bottom layer 2 mm thick as well as in the top layer 2 mm thick exhibiting pore size of 150 m and adhering to the well walls. Even after swelling, the substrate remains attached to the well bottom without detaching and floating. This substrate is, thus, suitable for seeding and culturing broad range of cell types and has already been subject to in-vitro and in-vivo tests. Porosity in the bottom layer was equal to 98.5% and 98% for the top layer. Porosity was determined by image analysis of the 3D morphology of the freeze dried substrate obtained employing the microCT computer tomograph (for example Nano3DX Rigaku, Japan). The porosities shown above are the average values calculated from 5 repetitive measurements performed for randomly selected wells from the wellplate. Standard deviation of the measurements was less than 10% of the average value.

Example 18

[0148] Wells in a 96-well plate were layer by layer filled with a biocompatible material in the following sequence: [0149] 1. Ultrapure water (Milipore, purity II) [0150] 2. Layer of homogeneous collagen solution, concentration of 2 wt. % in ultrapure water [0151] 3. Layer of 2 wt. % homogeneous solution of chitosan in ultrapure water [0152] 4. Ultrapure water (Milipore, purity II).

[0153] The materials in the wellplate were frozen under controlled conditions after deposition of each layer first at 18 C. (1 hour), then at 35 C. (1 hour), and, finally, freeze dried at the condensator temperature of 95 C. and pressure lower than 1000 Pa to constant weight. Following freeze drying, collagen is cross-linked using EDC/NHS system directly in the well and, again, freeze dried under the same conditions as described above. The wells contain homogeneous 3D porous substrate with the total thickness of 5 mm exhibiting controlled pore size of 400 m in the bottom layer 2.5 mm thick as well as in the top layer 2.5 mm thick exhibiting pore size of 380 m and adhering to the well walls. Even after swelling, the substrate remains attached to the well bottom without detaching and floating. This substrate is, thus, suitable for seeding and culturing broad range of cell types and has already been subject to in-vitro and in-vivo tests. Porosity in the bottom layer was equal to 99.5% and 98% for the top layer. Porosity was determined by image analysis of the 3D morphology of the freeze dried substrate obtained employing the microCT computer tomograph (for example Nano3DX Rigaku, Japan). The porosities shown above are the average values calculated from 5 repetitive measurements performed for randomly selected wells from the wellplate. Standard deviation of the measurements was less than 10% of the average value.

Example 19

[0154] Wells in a 96-well plate were layer by layer filled with a biocompatible material in the following sequence: [0155] 1. Ultrapure water (Milipore, purity II) [0156] 2. Layer of homogeneous collagen solution, concentration of 2 wt. % in ultrapure water [0157] 3. Layer of 2 wt. % homogeneous suspension of silk fibroin in ultrapure water [0158] 4. Ultrapure water (Milipore, purity II).

[0159] The materials in the wellplate were frozen under controlled conditions after deposition of each layer first at 18 C. (1 hour), then at 35 C. (1 hour), and, finally, freeze dried at the condensator temperature of 95 C. and pressure lower than 1000 Pa to constant weight. Following freeze drying, collagen is cross-linked using EDC/NHS system directly in the well and, again, freeze dried under the same conditions as described above. The wells contain homogeneous 3D porous substrate with the total thickness of 4 mm exhibiting controlled pore size of 400 m in the bottom layer 2.5 mm thick as well as in the top layer 1.5 mm thick exhibiting pore size of 380 m and adhering to the well walls. Even after swelling, the scaffold remains attached to the well bottom without detaching and floating. This substrate is, thus, suitable for seeding and culturing broad range of cell types and has already been subject to in-vitro and in-vivo tests. Porosity in the bottom layer was equal to 99.5% and 97.6% for the top layer. Porosity was determined by image analysis of the 3D morphology of the freeze dried substrate obtained employing the microCT computer tomograph (for example Nano3DX Rigaku, Japan). The porosities shown above are the average values calculated from 5 repetitive measurements performed for randomly selected wells from the wellplate. Standard deviation of the measurements was less than 10% of the average value.