Automated tissue engineering system

• US 20060141623A1
• Filed: 04/08/2003
• Published: 06/29/2006
• Est. Priority Date: 04/08/2002
• Status: Active Grant

• Current Assignee: Octane Biotech Inc. (Lonza Group AG), Millenium Biologix Technologies Inc. (Millenium Biologix Corporation)
• Original Assignee: Millenium Biologix Inc (Millenium Biologix Corporation)
• Inventors: Smith, Timothy J. N., Rupert, Hagg, Roberto, Tommasini, Sydney, M Pugh, Kowell, D Misener, Yves, Larcher
• Granted Patent: US 8,492,140 B2
• Time in Patent Office: Days
• US Class Current: 435/383
• 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 23/28 disposable or single use
• CPC Class Codes: C12M 23/42 Integrated assemblies, e.g. cassettes or cartridges
• CPC Class Codes: C12M 23/52 Mobile; Means for transporting the apparatus transportable laboratories B01L1/52
• CPC Class Codes: C12M 25/14 Scaffolds; Matrices in general C12N5/0068
• CPC Class Codes: C12M 25/16 Particles; Beads; Granular material; Encapsulation chemical or physical processes conducted in the presence of fluids and solid particles B01J8/00
• CPC Class Codes: C12M 29/00 Means for introduction, extraction or recirculation of materials, e.g. pumps pumps per se F04B
• CPC Class Codes: C12M 29/04 Filters; Permeable or porous membranes or plates, e.g. dialysis
• CPC Class Codes: C12M 41/46 of cellular or enzymatic activity or functionality, e.g. cell viability
• CPC Class Codes: C12M 41/48 Automatic or computerized control automatic analysis G01N35/00
• CPC Class Codes: C12M 45/09 by enzymatic treatment
• CPC Class Codes: C12M 47/06 Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material lysis of microorganisms C12N1/06; extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: C12M 47/12 Purification C12M47/04 takes precedence
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/0618 Cells of the nervous system
• CPC Class Codes: C12N 5/0654 Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
• CPC Class Codes: C12N 5/0655 Chondrocytes; Cartilage

Abstract

The invention provides systems, modules, bioreactor and methods for the automated culture, proliferation, differentiation, production and maintenance of tissue engineered products. In one aspect is an automated tissue engineering system comprising a housing, at least one bioreactor supported by the housing, the bioreactor facilitating physiological cellular functions and/or the generation of one or more tissue constructs from cell and/or tissue sources. A fluid containment system is supported by the housing and is in fluid communication with the bioreactor. One or more sensors are associated with one or more of the housing, bioreactor or fluid containment system for monitoring parameters related to the physiological cellular functions and/or generation of tissue constructs; and a microprocessor linked to one or more of the sensors. The systems, methods and products of the invention find use in various clinical and laboratory settings.

Claims

1-137. -137. (canceled)

138. An automated tissue engineering system comprising;

a housing;

at least one bioreactor supported by said housing, said bioreactor facilitating physiological cellular functions and/ or the generation of one or more tissue constructs from cell and/or tissue sources;

a fluid containment system supported by said housing and in fluid communication with said bioreactor,

one or more sensors associated with one or more of said housing, bioreactor or fluid containment system for monitoring parameters related to said physiological cellular functions and/or generation of tissue constructs; and

a microprocessor linked to one or more of said sensors.

139. The system of claim 138, wherein said bioreactor comprises a bioreactor housing;

one or more inlet ports and one or more outlet ports for media flow; and

at least one chamber defined within said bioreactor housing for receiving a variety of cells and/or tissues.

140. The system of claim 139, wherein said bioreactor housing comprises a lid, said one or more inlet ports and outlet ports being provided within said bioreactor housing and/or said lid.

141. The system of claim 138, wherein said chamber is selected from the group consisting of a tissue digestion chamber, culture/proliferation chamber, differentiation/tissue formation chamber and combinations thereof.

142. The system of claim 139, wherein two or more chambers are provided operably connected within said bioreactor.

143. The system of claim 139, wherein said chamber houses one or more substrates and/or scaffolds.

144. The system of claim 143, wherein said substrate is a contained suspension of micro-carrier.

145. The system of claims 139, wherein said chamber contains a plurality of zones.

146. The system of claim 145, wherein said plurality of zones may each accommodate a scaffold and/or substrate.

147. The system of claim 142, wherein at least one of said two or more chambers and said bioreactors are one of at least independently operable and co-operatively operable.

148. The system of claim 142, wherein at least one of said chambers and bioreactors are operably connected to provide for the exchange of one or more of fluids, cells and tissues between at least one of said chambers and said bioreactors.

149. The system of claim 148, wherein at least one of said chambers and said bioreactor are connected via at least one of a passageway, tubing, connector, valve, pump, filter, fluid access port, in-line gas exchange membrane, in-line sensor and void in a separation wall.

150. The system of claim 138, wherein said bioreactor is removably accomodated with said fluid containment system via said inlet port and said outlet port.

151. An automated tissue engineering system comprising;

a housing;

at least one tissue engineering module removably accommodated within said housing, said tissue engineering module comprising a support structure that holds at least one bioreactor, said bioreactor facilitating at least one of cell culture and tissue engineering functions, a fluid containment system in fluid communication with said bioreactor, and one or more sensors for monitoring parameters related to at least one of said cell culture and tissue engineering functions; and

a microprocessor disposed in said housing and linked to said tissue engineering module, said microprocessor controlling the operation of said tissue engineering module.

152. The tissue engineering system of claim 151, wherein said bioreactor comprises

a bioreactor housing having one or more inlet ports and one or more outlet ports for media flow;

and

at least one chamber defined within said bioreactor housing for receiving at least one of said cells and tissues and facilitating said cell culture and tissue engineering functions.

153. The tissue engineering system of claim 152, wherein said chamber houses one or more substrates and/or scaffolds.

154. The tissue engineering system of claim 152, wherein at least one of, said two or more chambers, and said bioreactors are at least one of independently operable and cooperatively operable.

155. The tissue engineering system of claim 151, wherein at least one of said chambers and said bioreactors are operably connected to provide for the exchange of at least one of said fluids, cells and tissues between at least one of said chambers and said bioreactors.

156. The tissue engineering system of claim 153, wherein said chamber contains a plurality of zones to contain a plurality of substrates and/ or scaffolds.

157. The tissue engineering system of claim 151, wherein said fluid containment system comprises a plurality of flexible reservoirs connected by flexible tubing for supplying and retrieving fluid to and from said bioreactor.

158. The tissue engineering system of claim 151, wherein said housing comprises one or more environmental sensors and an environmental control unit to maintain said environmental conditions within one or more of said housing, tissue engineering module, bioreactor, fluid containment system, and said flexible reservoirs.

159. The tissue engineering system of claim 151, wherein said housing has at least one insertion slot for insertion of said tissue engineering module via at least one set of guides for receiving said support structure.

160. The tissue engineering system of claim 159, wherein said insertion slot and said guides are vertically or horizontally orientated with respect to said housing.

161. The tissue engineering system of claim 159, wherein said insertion slot has a movable door and a locking mechanism.

162. The tissue engineering system of claim 151, wherein said system additionally comprises a user interface in operation with said microprocessor, said user interface providing for the entry of user inputs to said microprocessor and/or the output of system status.

163. The tissue engineering system of claim 162, further comprising a data system to permit input, output, recording, transfer and storage of information.

164. The tissue engineering system of claim 163, further comprising a computer and communications link.

165. The tissue engineering system of claim 151, wherein said microprocessor performs quality control assessments of said cell culture and tissue engineering functions.

166. The tissue engineering system of claim 151, wherein said tissue engineering module additionally comprises one or more microprocessors that are in operable connection with said microprocessor disposed in said housing.

167. A portable and sterilizable tissue engineering module, the module comprising;

a structural support holding at least one bioreactor, said bioreactor facilitating cell culture and tissue engineering functions;

a fluid containment system in fluid communication with said bioreactor; and

one or more sensors for monitoring parameters related to said cell culture and tissue engineering functions.

168. The tissue engineering module of claim 167, wherein said bioreactor comprises:

a bioreactor housing having one or more inlet ports and one or more outlet ports for media flow; and

at least one chamber defined within said bioreactor housing for receiving at least one of said cells and tissues and facilitating said cell culture and tissue engineering functions.

169. The tissue engineering module of claim 168, wherein said chamber houses one or more substrates and/or scaffolds.

170. The tissue engineering module of claim 168, wherein two or more chambers are provided operably connected within said bioreactor.

171. The tissue engineering module of claim 167, wherein two or more bioreactors are operably connected.

172. The tissue engineering module of claim 170, wherein at least one of said two or more chambers, and said bioreactors are independently operable and/or co-operatively operable.

173. The tissue engineering module of claim 172, wherein said module comprises a first bioreactor, a second bioreactor and a third bioreactor, said first bioreactor containing a tissue digestion chamber, said second bioreactor containing a culture/ proliferation chamber, said third bioreactor containing a differentiation/tissue formation chamber, and wherein said first, second and third bioreactors are operatively connected.

174. The tissue engineering module of claim 170, wherein said chambers and/or bioreactors are operably connected to provide for the exchange of fluids, cells and/or tissues between said chambers and/or bioreactors.

175. The tissue engineering module of claim 168, wherein said chamber contains a plurality of zones to contain a plurality of substrates and/or scaffolds.

176. The tissue engineering module of claim 167, wherein said fluid containment system comprises a plurality of flexible reservoirs connected by flexible tubing for supplying and retrieving fluid to and from said bioreactor.

177. The tissue engineering module of claim 176, wherein at least one of said flexible reservoir and said tubing is provided with fluid access ports for the loading or removal of material from said fluid containment system.

178. The tissue engineering module of claim 167, wherein a plurality of fluid flow control valves are provided in operable connection with fluid containment system to control the flow of fluid with said fluid containment system and said bioreactor.

179. The tissue engineering module of claim 178, wherein said module additionally comprises one or more pump units in connection with said fluid containment system for the pumping of fluid throughout said fluid containment system.

180. The tissue engineering module of claim 167, wherein a fluid flow plate is mounted or provided integral to said structural support, said fluid flow plate being operationally connected to said fluid control valves to direct fluid flow within and between said fluid containment system and said bioreactor.

181. The tissue engineering module of claim 167, wherein said tissue engineering module additionally comprises a heating and mixing chamber to heat and mix fluids flowing to said bioreactor.

182. The tissue engineering module of claim 167, wherein one or more gas exchange membranes are provided within one of or between said fluid containment system and bioreactor, said gas exchange membranes permitting the transfer of gaseous products into or out of fluid flowing to or resident in said bioreactor.

183. The tissue engineering module of claim 167, wherein said module additionally comprises a thermoelectric element in operable connection with said bioreactor.

184. The tissue engineering module of claim 167, wherein said module additionally comprises one or more microprocessors in operable connection with said sensors.

185. The tissue engineering module of claim 167, wherein said module additionally comprises one or more access ports, said access ports being operatively linked with at least one of said bioreactor and said fluid containment system, said access ports providing for sterile loading or removal of cells, fluids, cell and tissue culture media, growth factors, pharmaceutical agents, quality control reagents, quality control samples and other materials.

186. The tissue engineering module of claim 185, wherein said access ports are operatively linked to a syringe manifold integrated with said structural support.

187. The tissue engineering module of claim 167, wherein said bioreactor is integrally mounted to said structural support.

188. The tissue engineering module of claim 167, wherein said bioreactor is detachable from said structural support.

189. The tissue engineering module of claim 167, wherein a camera is provided for visual inspection within said bioreactor.

190. The tissue engineering module of claim 167, wherein said module additionally comprises an identifying element.

191. The tissue engineering module of claim 167, wherein said module is sterilizable before use and disposable after use.

192. A bioreactor for facilitating and supporting cellular functions and/or the generation of tissue constructs, said bioreactor comprising;

a bioreactor housing;

one or more inlet ports and one or more outlet ports for media flow;

at least one chamber defined within said bioreactor housing for facilitating and supporting cellular functions and/or the generation of one or more tissue constructs from cell and/ or tissue sources; and

one or more sensors for monitoring parameters related to said cellular functions and/or generation of tissue constructs within said at least one chamber.

193. The bioreactor of claim 192, wherein said bioreactor housing comprises a lid, said lid.

194. The bioreactor of claim 192, wherein said bioreactor comprises a single culture/proliferation chamber having at least one of a culture/proliferation scaffold and a substrate supported therein.

195. The bioreactor of claim 192, wherein said bioreactor comprises a culture/proliferation chamber connected to a downstream differentiation/tissue formation chamber, said culture/proliferation chamber having at least one of a proliferation scaffold and a substrate supported therein, said differentiation/ tissue formation chamber having an implantable differentiation scaffold supported therein.

196. The bioreactor of claim 192, wherein a connecting passageway is provided between said culture/proliferation chamber and said differentiation/tissue formation chamber, said connecting passageway facilitating movement of cells released from said proliferation scaffold and/or substrate to said differentiation scaffold.

197. The bioreactor of claim 196, further comprising a digestion chamber for the digestion of tissue biopsy material, said digestion chamber being upstream of said culture/proliferation chamber.

198. The bioreactor of claim 195, wherein one or more filters are provided at a location selected from upstream of said proliferation scaffold, upstream of said differentiation scaffold, upstream to said outlet port and combinations thereof.

199. The bioreactor of claim 196, wherein said differentiation/tissue formation chamber contains a plurality of zones to contain a plurality of differentiation scaffolds and/ or substrates.

200. The bioreactor of claim 192, wherein said bioreactor has a sampling port that is operatively connected to one or more of said chambers.

201. The bioreactor of claim 192, wherein a gas exchange membrane forms part of said chamber.

202. The bioreactor of claim 192, wherein said bioreactor is removably accomodated with a fluid containment system via said inlet port and said outlet port.

203. The bioreactor of claim 192, wherein a mixing diaphragm forms part of said chamber and is operably connected to at least one of a mixing actuator and a mixing drive.

204. The bioreactor of claim 203, wherein said mixing diaphragm is incorporated within said bioreactor.

205. The bioreactor of claim 192, wherein said bioreactor is operatively connected to at least one of an impact actuator and an impact drive.

206. The bioreactor of claim 192, wherein said chamber further supports physiological stimulation of resident cells and/or tissues.

207. The bioreactor of claim 192, further comprising a micro-loading diaphragm in operable connection with at least one of a micro-loading actuator and a micro-loading drive.

208. The bioreactor of claim 206, wherein said stimulation is performed by supplying an electric field.

209. The bioreactor of claim 192, wherein said bioreactor is operationally accommodated within a tissue engineering module comprising a fluid containment system affixed to a structural support and in fluid communication with said bioreactor, and wherein said fluid containment system comprises a plurality of flexible reservoirs connected by flexible tubing for supplying and retrieving fluid to and from said bioreactor.

210. The bioreactor of claim 209, wherein said tissue engineering module is accommodated within a housing of an automated tissue engineering system, said system comprising a microprocessor for operation with said module and wherein said microprocessor controls the functioning of said module.

211. The bioreactor of claim 192, wherein said bioreactor additionally comprises an optical probe.

212. The bioreactor of claim 192, wherein said bioreactor is sterilizable before use and disposable after use.

213. A method for the automated digestion of a tissue biopsy, the method comprising;

loading a tissue biopsy within a bioreactor connected with a media reservoir and flow system, said bioreactor having one or more sensors to detect physiological conditions within said bioreactor for assessment by a microprocessor;

providing tissue digestion enzymes within said bioreactor; and

monitoring and maintaining digestion conditions within said bioreactor for a sufficient period of time for a desired level of tissue digestion.

214. A method for the automated proliferation of cells, said method comprising;

seeding cells onto a proliferation substrate or scaffold supported within a bioreactor connected with a media reservoir and flow system, said bioreactor having one or more sensors to detect physiological conditions within said bioreactor for assessment by a microprocessor; and

monitoring and maintaining suitable culturing conditions within said bioreactor for a sufficient period of time for a desired level of cell proliferation.

215. A method for the automated differentiation of cells, said method comprising;

seeding cells onto a differentiation substrate or scaffold supported within a bioreactor connected with a media reservoir and flow system, said bioreactor having one or more sensors to detect physiological conditions within said bioreactor for assessment by a microprocessor; and

monitoring and maintaining suitable culturing conditions within said bioreactor for a sufficient period of time for a desired level of cell differentiation.

216. A method for the production of a tissue construct, said method comprising;

seeding cells onto a scaffold supported within a bioreactor, said bioreactor connected with a media reservoir and flow system, said bioreactor having one or more sensors to detect physiological conditions within said bioreactor to a microprocessor, and

monitoring and maintaining suitable culturing conditions within said bioreactor for a sufficient period of time for said cells to express extracellular matrix that provides structural support for the tissue construct.

217. An automated method for digesting a tissue biopsy to provide primary cells, including precursor cells, and further proliferating and differentiating the cells to enable the formation of a tissue implant, the method comprising;

loading a tissue biopsy within a bioreactor connected with a media reservoir and flow system, said bioreactor having one or more sensors to detect physiological conditions within said bioreactor for assessment by a microprocessor;

providing tissue digestion enzymes;

monitoring and maintaining suitable digestion conditions within said bioreactor for a sufficient period of time to obtain disassociated cells;

seeding the disassociated cells onto a proliferation substrate or scaffold supported within a bioreactor connected with a media reservoir and flow system, said bioreactor having one or, more sensors to detect physiological conditions within said bioreactor for assessment by a microprocessor,

monitoring and maintaining suitable culturing conditions within said bioreactor for a sufficient period of time to obtain the desired level of cell proliferation and expansion;

releasing the expanded cells from the proliferation substrate or scaffold;

seeding the expanded cells onto a differentiation substrate or scaffold supported within a bioreactor connected with a media reservoir and flow system, said bioreactor having one or more sensors to detect physiological conditions within said bioreactor for assessment by a microprocessor, and

monitoring and maintaining suitable culturing conditions within said bioreactor for a sufficient period of time to obtain a tissue implant.

218. A method for providing a skeletal implant, the method comprising;

seeding osteogenic and/ or osteoprogenitor cells onto a porous scaffold of a bone biomaterial supported within a bioreactor connected with a media reservoir and flow system, said bioreactor having one or more sensors to detect physiological conditions within said bioreactor for assessment by a microprocessor; and

monitoring and maintaining suitable conditions within said bioreactor for a sufficient period of time to allow the osteogenic and/ or osteoprogenitor cells to proliferate and/or differentiate throughout the scaffold to provide a tissue implant for orthopedic applications.

219. A method for providing a cartilage implant, the method comprising;

seeding chondrogenic and/or chondroprogenitor cells onto a porous scaffold of a biomaterial supported within a bioreactor connected with a media reservoir and flow system, said bioreactor having one or more sensors to detect physiological conditions within said bioreactor for assessment by a microprocessor; and

monitoring and maintaining suitable conditions within said bioreactor for a sufficient period of time to allow the chondrogenic and/or chondroprogenitor cells to proliferate and/or differentiate throughout the scaffold to provide a cartilage implant.

220. A method for providing an implant for reestablishing the inner nucleus of a spinal disc, the method comprising;

seeding nucleus pulposus cells within a scaffold a porous scaffold of a biomaterial supported within a bioreactor connected with a media reservoir and flow system, said bioreactor having one or more sensors to detect physiological conditions within said bioreactor to a microprocessor; and

monitoring and maintaining suitable conditions within said bioreactor for a sufficient period of time to allow proliferation and/or differentiation of the nucleus pulposus cells and the expression of extracellular matrix components characteristic of the nucleus pulposus.

221. A method for washing cells, said method comprising:

loading a cell suspension containing one or more undesired chemicals into a chamber,

continuously recirculating the cell suspension from the chamber through a cross-flow filtration module that comprises a membrane impermeable to said cells but permeable to said undesired chemicals to provide a washed cell suspension; and

collecting the washed cell suspension.

222. A method for enrichment of cells, said method comprising:

loading a cell suspension containing excessive cell suspension volume into a chamber;

continuously recirculating the cell suspension from the chamber through a cross-flow filtration module that comprises a membrane impermeable to the cells but allowing the excessive cell suspension volume to be removed and collected.