FABRICATION OF VASCULARIZED TISSUE USING MICROFABRICATED TWO-DIMENSIONAL MOLDS

• US 20100267136A1
• Filed: 05/24/2010
• Published: 10/21/2010
• Est. Priority Date: 04/30/1999
• Status: Active Grant

• Current Assignee: Charles Stark Draper Laboratory Incorporated, The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: Charles Stark Draper Laboratory Incorporated, The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Ishii, Osamu, Ogilvie, Jennifer, Sevy, Alexander, King, Kevin R., Shin, Young-Moon Michael, Maemura, Tomoyuki, Wang, Chiao-Chun, Borenstein, Jeffrey T., Kaazempur-Mofrad, Mohammad Reza, Vacanti, Joseph P., Weinberg, Eli
• Granted Patent: US 8,642,336 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A61F 2/062 Apparatus for the production of blood vessels made from natural tissue or with layers of living cells prostheses made from natural tissue or living cells A61L27/507
• CPC Class Codes: A61F 2240/004 Using a positive or negative model, e.g. moulds
• CPC Class Codes: A61F 2240/005 Templates
• CPC Class Codes: A61L 2300/64 Animal cells
• CPC Class Codes: A61L 27/3604 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
• CPC Class Codes: A61L 27/3804 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
• CPC Class Codes: A61L 27/3808 Endothelial cells
• CPC Class Codes: A61L 27/3891 as distinct cell layers
• CPC Class Codes: A61L 27/3895 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
• CPC Class Codes: A61L 27/58 Materials at least partially resorbable by the body
• CPC Class Codes: B33Y 80/00 Products made by additive manufacturing
• CPC Class Codes: C12M 21/08 for producing artificial tissue or for ex-vivo cultivation of tissue prostheses A61F2/00, grafts A61L27/00
• CPC Class Codes: C12M 23/06 Tubular C12M23/08, C12M23/16 take precedence
• CPC Class Codes: C12M 23/30 biodegradable
• CPC Class Codes: C12M 25/02 Membranes; Filters filters or filtration in general B01D24/00-B01D41/00
• CPC Class Codes: C12M 35/08 Chemical, biochemical or biological means, e.g. plasma jet, co-culture
• CPC Class Codes: C12N 2533/12 Glass
• CPC Class Codes: C12N 2533/30 Synthetic polymers thermoreactive polymers, e.g. PNIPAm, C12N2539/10
• CPC Class Codes: C12N 5/0062 General methods for three-dimensional culture
• CPC Class Codes: C12N 5/0697 Artificial constructs associating cells of different lineages, e.g. tissue equivalents blood vessels C12N5/0691
• CPC Class Codes: Y10T 29/49826 : Assembling or joining

Abstract

Methods and materials for making complex, living, vascularized tissues for organ and tissue replacement, especially complex and/or thick structures, such as liver tissue is provided. Tissue lamina is made in a system comprising an apparatus having (a) a first mold or polymer scaffold, a semi-permeable membrane, and a second mold or polymer scaffold, wherein the semi-permeable membrane is disposed between the first and second molds or polymer scaffolds, wherein the first and second molds or polymer scaffolds have means defining microchannels positioned toward the semi-permeable membrane, wherein the first and second molds or polymer scaffolds are fastened together; and (b) animal cells. Methods for producing complex, three-dimensional tissues or organs from tissue lamina are also provided.

Claims

1. An apparatus for making tissue lamina comprising a first mold, a semi-permeable membrane, and a second mold, wherein the semi-permeable membrane is disposed between the first and second molds, wherein the first and second molds have means defining microchannels positioned toward the semi-permeable membrane, wherein the first and second molds are fastened together, and wherein each of the first and second molds and the semi-permeable membrane are comprised of material that is suitable for attachment and culturing of animal cells.

2. The apparatus of claim 1, further comprising nutrient supply and excretion removal lines in fluid communication with the apparatus.

3. The apparatus of claim 2, further comprising pumping means for circulating fluid through the apparatus.

4. The apparatus of claim 1, further comprising a coating on the microchannels that promotes cell adhesion to the apparatus.

5. The apparatus of claim 1, further comprising a coating on the microchannels that promotes lifting of cells as intact sheets from the apparatus.

6-27. -27. (canceled)

28. A system for making tissue lamina comprising:

(a) an apparatus for making tissue lamina comprising a first mold, a semi-permeable membrane, and a second mold, wherein the semi-permeable membrane is disposed between the first and second molds, wherein the first and second molds have means defining microchannels positioned toward the semi-permeable membrane, wherein the first and second molds are fastened together, and wherein each of the first and second molds and the semi-permeable membrane are comprised of material that is suitable for attachment and culturing of animal cells; and

(b) animal cells.

29. The system of claim 28, further comprising nutrient supply and excretion removal lines in fluid communication with the apparatus.

30. The system of claim 28, further comprising pumping means for circulating fluid through the apparatus.

31. The system of claim 28, further comprising a coating on the microchannels that promotes cell adhesion to the apparatus.

32-52. -52. (canceled)

53. A method of making an apparatus for making tissue lamina comprising the steps of:

(a) positioning a semi-permeable membrane between a first and second mold, wherein the first and second molds have means defining microchannels positioned toward the semi-permeable membrane, and wherein each of the first and second molds and the semi-permeable membrane are comprised of material that is suitable for attachment and culturing of animal cells; and

(b) fastening the first and second molds together such that the semi-permeable membrane is disposed between the molds.

54. The method of claim 53, further comprising the step of connecting nutrient supply and excretion removal lines in fluid communication with the apparatus.

55. The method of claim 53, wherein the microchannels are coated with a material that promotes cell adhesion to the apparatus.

56-76. -76. (canceled)

77. A method of making tissue lamina comprising the steps of:

(a) positioning a semi-permeable membrane between a first and second mold, wherein the first and second molds have means defining microchannels positioned toward the semi-permeable membrane, and wherein each of the first and second molds and the semi-permeable membrane are comprised of material that is suitable for attachment and culturing of animal cells;

(b) fastening the first and second molds together such that the semi-permeable membrane is disposed between the molds, thereby forming an apparatus; and

(c) culturing cells in the microchannels of the molds.

80. The method of claim 77, further comprising the step of

(d) connecting nutrient supply and excretion removal lines in fluid communication with the apparatus.

81. The method of claim 80, further comprising the step of

(e) circulating fluid through the apparatus by means of a pump.

82. The method of claim 77, wherein the microchannels are coated with a material that promotes cell adhesion to the apparatus.

104. A composition comprising tissue lamina obtained by the method of claim 77.

78. (canceled)

79. (canceled)

83-103. -103. (canceled)

105. A method of making multiple layers of tissue lamina in three dimensions comprising the steps of:

(a) positioning a semi-permeable membrane between a first and second mold, wherein the first and second molds have means defining microchannels positioned toward the semi-permeable membrane, and wherein each of the first and second molds and the semi-permeable membrane are comprised of material that is suitable for attachment and culturing of animal cells;

(b) fastening the first and second molds together such that the semi-permeable membrane is disposed between the molds, thereby forming an apparatus;

(c) culturing cells in the microchannels of the first and second molds to form tissue lamina;

(d) removing the tissue lamina from the first and second molds; and

(e) folding or rolling the tissue lamina to form multiple layers of tissue lamina in three dimensions.

112. A composition comprising multiple layers of tissue lamina obtained by the method of claim 105.

106-111. -111. (canceled)

113. A method of making multiple layers of tissue lamina in three dimensions comprising the steps of:

(a) positioning a semi-permeable membrane between a first and second mold, wherein the first and second molds have means defining microchannels positioned toward the semi-permeable membrane;

(b) fastening the first and second molds together such that the semi-permeable membrane is disposed between the molds;

(c) fastening a third mold adjacent to the first or second mold, wherein the third mold has means defining microchannels positioned toward the first or second mold, thereby forming an apparatus, wherein each of the first, second and third molds and the semi-permeable membrane are comprised of material that is suitable for attachment and culturing of animal cells; and

(d) culturing cells in the microchannels of the first, second and third molds to form tissue lamina.

117. A composition comprising multiple layers of tissue lamina obtained by the method of claim 113.

114-116. -116. (canceled)

118. (canceled)

119. An apparatus for making tissue lamina, comprising:

(a) a first polymer scaffold, a semi-permeable membrane, and a second polymer scaffold, wherein the semi-permeable membrane is disposed between the first and second polymer scaffolds, wherein the first and second polymer scaffolds have means defining microchannels positioned toward the semi-permeable membrane, wherein the first and second polymer scaffolds are fastened together, and wherein each of the first and second polymer scaffolds and the semi-permeable membrane are comprised of material that is suitable for attachment and culturing of animal cells; and

(b) nutrient supply and excretion removal lines in fluid communication with the apparatus.

120. The apparatus of claim 119, further comprising pumping means for circulating fluid through the apparatus.

121. The apparatus of claim 119, further comprising a coating on the microchannels that promotes cell adhesion to the apparatus.

122-144. -144. (canceled)

145. A system for making tissue lamina comprising:

(a) an apparatus for making tissue lamina comprising a first polymer scaffold, a semi-permeable membrane, and a second polymer scaffold, wherein the semi-permeable membrane is disposed between the first and second polymer scaffolds, wherein the first and second polymer scaffolds have means defining microchannels positioned toward the semi-permeable membrane, wherein the first and second polymer scaffolds are fastened together, and wherein each of the first and second polymer scaffolds and the semi-permeable membrane are comprised of material that is suitable for attachment and culturing of animal cells;

(b) animal cells; and

(c) nutrient supply and excretion removal lines in fluid communication with the apparatus.

146. The system of claim 145, further comprising pumping means for circulating fluid through the apparatus.

147. The system of claim 145, further comprising a coating on the microchannels that promotes cell adhesion to the apparatus.

148-168. -168. (canceled)

169. A method of making an apparatus for making tissue lamina comprising the steps of:

(a) positioning a semi-permeable membrane between a first and second polymer scaffold, wherein the first and second polymer scaffolds have means defining microchannels positioned toward the semi-permeable membrane, and wherein each of the first and second polymer scaffolds and the semi-permeable membrane are comprised of material that is suitable for attachment and culturing of animal cells;

(b) fastening the first and second polymer scaffolds together such that the semi-permeable membrane is disposed between the polymer scaffolds; and

(c) connecting nutrient supply and excretion removal lines in fluid communication with the apparatus.

170. The method of claim 169, wherein the microchannels are coated with a material that promotes cell adhesion to the apparatus.

171-193. -193. (canceled)

194. A method of making tissue lamina comprising the steps of

(a) positioning a semi-permeable membrane between a first and second polymer scaffold, wherein the first and second polymer scaffolds have means defining microchannels positioned toward the semi-permeable membrane, and wherein each of the first and second polymer scaffolds and the semi-permeable membrane are comprised of material that is suitable for attachment and culturing of animal cells;

(b) fastening the first and second polymer scaffolds together such that the semi-permeable membrane is disposed between the polymer scaffolds, thereby forming an apparatus;

(c) culturing cells in the microchannels of the polymer scaffolds; and

(d) connecting nutrient supply and excretion removal lines in fluid communication with the apparatus.

196. The method of claim 194, wherein the microchannels are coated with a material that promotes cell adhesion to the apparatus.

195. (canceled)

197-217. -217. (canceled)

218. A method of making multiple layers of tissue lamina in three dimensions comprising the steps of:

(a) positioning a semi-permeable membrane between a first and second polymer scaffold, wherein the first and second polymer scaffolds have means defining microchannels positioned toward the semi-permeable membrane, and wherein each of the first and second polymer scaffolds and the semi-permeable membrane are comprised of material that is suitable for attachment and culturing of animal cells;

(b) fastening the first and second polymer scaffolds together such that the semi-permeable membrane is disposed between the polymer scaffolds, thereby forming an apparatus;

(c) culturing cells in the microchannels of the first and second polymer scaffolds to form tissue lamina;

(d) removing the tissue lamina from the first and second polymer scaffolds; and

(e) folding or rolling the tissue lamina to form multiple layers of tissue lamina in three dimensions.

225. A composition comprising multiple layers of tissue lamina obtained by the method of claim 218.

219-224. -224. (canceled)

226. A method of making multiple layers of tissue lamina in three dimensions comprising the steps of:

(a) positioning a semi-permeable membrane between a first and second polymer scaffold, wherein the first and second polymer scaffolds have means defining microchannels positioned toward the semi-permeable membrane;

(b) fastening the first and second polymer scaffolds together such that the semi-permeable membrane is disposed between the polymer scaffolds;

(c) fastening a third polymer scaffold adjacent to the first or second polymer scaffold, wherein the third polymer scaffold has means defining microchannels positioned toward the first or second polymer scaffold, thereby forming an apparatus, wherein each of the first, second and third polymer scaffolds and the semi-permeable membrane are comprised of material that is suitable for attachment and culturing of animal cells; and

(d) culturing cells in the microchannels of the first, second and third polymer scaffolds to form tissue lamina.

230. A composition comprising multiple layers of tissue lamina obtained by the method of claim 226.

227-229. -229. (canceled)

231-268. -268. (canceled)

269. An apparatus for forming vascularized tissue, comprising:

(a) a first substrate having a tissue-defining surface comprising a pattern of open-faced channels defined by walls extending from the tissue-defining surface into the first substrate;

(b) a second substrate having a tissue-defining surface comprising at least one open-faced compartment defined by walls extending from the tissue-defining surface into the second substrate; and

(c) a semi-permeable membrane characterized by a high permeability for nutrients and a low permeability for animal cells;

wherein the first substrate, the second substrate, and the semi-permeable membrane are arranged in a sandwich configuration with the tissue-defining surface of the first substrate being disposed opposite to the tissue-defining surface of the second substrate, and with the semi-permeable membrane being disposed between the tissue-defining surfaces of the first and second substrates;

wherein the walls defining the channels and the compartment(s) are suitable for attachment and culturing of animal cells; and

wherein have diameter of the channels is between about 1 to 500 μ

m.

270. (canceled)

271. (canceled)