Devices and methods for pharmacokinetic-based cell culture system

• US 20030082795A1
• Filed: 04/25/2002
• Published: 05/01/2003
• Est. Priority Date: 04/25/2001
• Status: Active Grant

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Inventors: Harrison, Robert Andrew, Shuler, Michael, Meyers, Scott, Baxter, Gregory T., Sin, Aaron
• Granted Patent: US 7,288,405 B2
• Time in Patent Office: Days
• US Class Current: 435/286.1
• CPC Class Codes: C12M 23/16 Microfluidic devices; Capillary tubes integrated microfluidic structures B01L3/5027; microreactors B01J19/0093
• CPC Class Codes: C12M 23/44 Multiple separable units; Modules
• CPC Class Codes: C12M 35/08 Chemical, biochemical or biological means, e.g. plasma jet, co-culture
• 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: C12N 5/0062 General methods for three-dimensional culture
• CPC Class Codes: C12N 5/0671 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5067 Liver cells

Abstract

Devices, in vitro cell cultures, systems, and methods are provided for microscale cell culture analogous (CCA) device.

Claims

1. A pharmacokinetic-based microscale culture device, comprising:

a first microscale chamber containing a first type of cell under conditions where the first type of cell provides at least one pharmacokinetic parameter value comparable to a value obtained for the same type of cell in vivo, wherein the first chamber comprises a first inlet and a first outlet for flow of culture medium;

a second microscale chamber containing a second type of cell under conditions where the second type of cell provides at least one pharmacokinetic parameter value comparable to a value obtained for the same type of cell in vivo, wherein the second chamber comprises a second inlet and a second outlet for flow of culture medium; and

a microfluidic channel interconnecting the first and second chambers.

2. The culture device of claim 1, further comprising one or more additional microscale chambers containing an additional type of cell under conditions where the additional cell provides at least one pharmacokinetic parameter value comparable to a value obtained for the same type of cell in vivo, wherein the one or more additional chambers comprise an inlet and outlet for flow of culture medium.

3. The culture device of claim 1, further comprising culture medium.

4. The culture device of claim 3, wherein the culture medium flows through the chambers.

5. The culture device of claim 3, wherein the culture medium flows re-circulated through the chambers.

6. The culture device of claim 3, further comprising a pumping mechanism.

7. The culture device of claim 6, wherein the pumping mechanism is integrated in the device.

8. The culture device of claim 6, wherein the pumping mechanism is external to the device.

9. The culture device of claim 1, further comprising a debubbler located within the microfluidic channel.

10. The culture device of claim 1, further comprising a debubbler located externally to the device.

11. The culture device of claim 1, wherein at least one of the pharmacokinetic parameters is a measurement of interaction between cells, liquid residence time, liquid to cell ratio, metabolism by cells, or shear stress.

12. The culture device of claim 1, further comprising at least one sensor for obtaining signals from cultured cells.

13. The culture device of claim 12, wherein the at least one sensor is a biosensor.

14. The culture device of claim 12, wherein the at least one sensor comprises a waveguide.

15. The culture device of claim 1, wherein the device is microfabricated.

16. The culture device of claim 1, wherein the device is manufactured from a microfabricated master.

17. The culture device of claim 1, wherein at least one of the chambers provides for three-dimensional growth of cells.

18. The culture device of claim 1, wherein at least one of the chambers contains a plurality of cells.

19. The culture device of claim 1, wherein at least one of the chambers contains a tissue biopsy.

20. The culture device of claim 1, wherein at least one of the chambers contains a cross-section of a tissue.

21. The culture device of claim 19 or 20, wherein the tissue is healthy or diseased.

22. The culture device of claim 19 or 20, wherein the tissue is an artery, vein, gastrointestinal tract, esophagus, or colon.

23. The culture device of claim 1, wherein at least one of the chambers contains a cross-section of an organ.

24. The culture device of claim 23, wherein the organ is healthy or diseased.

25. The culture device of claim 23, wherein the organ is a heart, brain, kidney, lung, or muscle.

26. The culture device of claim 1, wherein at least one of the chambers contains circulating or adherent cells.

27. The culture device of claim 1, wherein at least one of the chambers contains eukaryotic cells.

28. The culture device of claim 27, wherein the eukaryotic cells are plant or animal cells.

29. The culture device of claim 28, wherein the cells are mammalian cells.

30. The culture device of claim 1, wherein at least one of the chambers contains prokaryotic cells.

31. The culture device of claim 1, wherein the cells are primary cells.

32. The culture device of claim 1, wherein the cells are tumor cells.

33. The culture device of claim 1, wherein the cells are stem cells.

34. The culture device of claim 1, wherein the cells are genetically altered, transformed or immortalized cells.

107. The method for producing a biological product comprising providing the culture device of claim 1 or 73 containing a culture medium, and a cell that generates the biological product.

108. The method of claim 107, wherein the cell is a host cell genetically engineered to produce an exogenous gene product.

109. The method of claim 107, wherein the biological product is a growth factor, regulatory factor, peptide hormone, or antibody.

110. The method of claim 107, further comprising isolating the biological product from the culture medium using a standard isolation technique.

111. The method of claim 110, wherein the isolation technique is HPLC, column chromatography, or electrophoresis.

35. A pharmacokinetic-based culture system of cells grown in a microscale culture device, comprising:

a first chamber comprising a first cell type maintained under conditions providing at least one pharmacokinetic parameter value comparable to values obtained for the cells in vivo;

a second chamber of different geometry than the first chamber comprising a second cell type maintained under conditions providing at least one pharmacokinetic parameter value comparable to values obtained for the cells in vivo;

wherein the first and second chambers are interconnected by fluidic channels; and

an inlet and outlet for recirculation of culture medium.

36. The culture system of claim 35, comprising multiple interconnected devices.

37. The culture system of claim 35, wherein the at least one pharmacokinetic parameter a measurement of interactions between cells, liquid residence time, liquid to cell ratios, metabolism by cells, and shear stress.

38. The culture system of claim 35, wherein each of the chambers provides for at least two pharmacokinetic parameter values comparable to values obtained for a cell of interest in vivo.

39. The culture system of claim 35, further comprising a pumping mechanism.

40. The culture system of claim 35, further comprising a debubbler located within a microfluidic channel.

41. The culture system of claim 35, further comprising at least one sensor for obtaining signals from cultured cells.

42. The culture system of claim 41, wherein the at least one sensor is a biosensor.

43. The culture system of claim 41, wherein the at least one sensor comprises a waveguide.

44. The culture system of claim 35, wherein the device is microfabricated.

45. The culture system of claim 35, wherein the device is manufactured from a microfabricated master.

46. The culture system of claim 35, wherein at least one of the chambers provides for three-dimensional growth of cells.

47. The culture system of claim 35, wherein at least one of the chambers contains a plurality of cells.

48. The culture system of claim 35, wherein at least one of the chambers contains a tissue biopsy.

49. The culture system of claim 35, wherein at least one of the chambers contains a cross-section of a tissue.

50. The culture system of claim 48 or 49, wherein the tissue is healthy or diseased.

51. The culture device of claim 48 or 49, wherein the tissue is an artery, vein, gastrointestinal tract, esophagus or colon.

52. The culture system of claim 35, wherein at least one of the chambers contains a cross-section of an organ.

53. The culture system of claim 52, wherein the organ is healthy or diseased.

54. The culture system of claim 48, wherein the organ is a heart, brain, kidney or lung.

55. The culture system of claim 35, wherein at least one of the chambers contains circulating or adherent cells.

56. The culture system of claim 35, wherein at least one of the chambers contains eukaryotic cells.

57. The culture system of claim 56, wherein the eukaryotic cells are plant or animal cells.

58. The culture system of claim 35, wherein at least one of the chambers contains prokaryotic cells.

59. The culture system of claim 57, wherein the cells are mammalian cells.

60. The culture system of claim 35, wherein the cells are primary cells.

61. The culture system of claim 35, wherein the cells are tumor cells.

62. The culture system of claim 35, wherein the cells are genetically altered, transformed or immortalized cells.

63. The culture system of claim 35, wherein the cells are stem cells.

64. A method for determining the effect of an input variable on a pharmacokinetic-based culture system of cells, the method comprising:

contacting the culture system of claim 35 with an input variable; and

monitoring at least one output parameter.

65. The method of claim 64, wherein the step of monitoring the at least one output parameter comprises obtaining information from at least one sensor in the device.

66. The method of claim 64, wherein the input variable is an organic compound.

67. The method of claim 64, wherein the input variable is an inorganic compound.

68. The method of claim 64, wherein the input variable is a complex sample.

69. The method of claim 64, wherein the input variable is a pharmaceutical, environmental sample, a nutritional sample, or a consumer product.

70. The method of claim 64, wherein the input variable is a virus, liposome, nanoparticle, biodegradable polymer, radiolabeled particle or toxin, biomolecul, toxin-conjugated particle or biomolecule.

71. The method of claim 64, wherein the input variable is a stabilizing agent.

72. The method of claim 71, wherein the stabilizing agent is albumin, polyethylene glycol, poly(ethylene-co-vinyl acetate), or poly(lactide-co-glycolide).

73. A pharmacokinetic-based microscale culture device, comprising:

a first microscale chamber containing a cell culture comprising a first type of cell under physiological conditions where the cell culture provides at least one pharmacokinetic parameter value comparable to a value obtained for the same type of cell culture in vivo, wherein the first chamber comprises a first inlet and a first outlet for flow of culture medium; and

a sensor for obtaining signals from the cell culture.

74. The culture device of claim 73, wherein the cell culture further comprises at least one additional type of cell.

75. The culture device of claim 73, further comprising:

a second microscale chamber containing a second type of cell culture under conditions where the second type of cell culture provides at least one pharmacokinetic parameter value comparable to a value obtained for the same type of cell in vivo, wherein the second chamber comprises a second inlet and a second outlet for flow of culture medium;

a sensor for obtaining signals from the cell culture; and

a microfluidic channel interconnecting the first and second chambers.

76. The culture device of claim 73, further comprising culture medium.

77. The culture device of claim 76, wherein the culture medium flows through the chambers.

78. The culture device of claim 76, wherein the culture medium flows re-circulated through the chamber.

79. The culture device of claim 73 further comprising a pumping mechanism.

80. The culture device of claim 79, wherein the pumping mechanism is integrated in the device.

81. The culture device of claim 79, wherein the pumping mechanism is external to the device.

82. The culture device of claim 73, further comprising a debubbler located within the microfluidic channel.

83. The culture device of claim 73, further comprising a debubbler located externally to the device.

84. The culture device of claim 73, wherein at least one of the pharmacokinetic parameters is a measurement of interaction between cells, liquid residence time, liquid to cell ratio, metabolism by cells, or shear stress.

85. The culture device of claim 73, wherein the sensor is a biosensor.

86. The culture device of claim 73, wherein the sensor comprises a waveguide.

87. The culture device of claim 73, wherein the device is microfabricated.

88. The culture device of claim 73, wherein the device is manufactured from a microfabricated master.

89. The culture device of claim 73, wherein the chamber provides for three-dimensional growth of cells.

90. The culture device of claim 73, wherein the chamber contains a plurality of cells.

91. The culture device of claim 73, wherein the chamber contains a tissue biopsy.

92. The culture device of claim 73, wherein the chamber contains a cross-section of a tissue.

93. The culture device of claim 91 or 92, wherein the tissue is healthy or diseased.

94. The culture device of claim 91 or 92, wherein the tissue is an artery, vein, gastrointestinal tract, esophagus, or colon.

95. The culture device of claim 73, wherein the chamber contains a cross-section of an organ.

96. The culture device of claim 95, wherein the organ is healthy or diseased.

97. The culture device of claim 95, wherein the organ is a heart, brain, kidney, lung, or muscle.

98. The culture device of claim 73, wherein the chamber contains circulating or adherent cells.

99. The culture device of claim 73, wherein the chamber contains eukaryotic cells.

100. The culture device of claim 99, wherein the eukaryotic cells are plant or animal cells.

101. The culture device of claim 100, wherein the cells are mammalian cells.

102. The culture device of claim 73, wherein the chamber contains prokaryotic cells.

103. The culture device of claim 73, wherein the cells are primary cells.

104. The culture device of claim 73, wherein the cells are tumor cells.

105. The culture device of claim 73, wherein the cells are stem cells.

106. The culture device of claim 73, wherein the cells are genetically altered, transformed, or immortalized cells.

112. A microscale culture device, comprising:

a first microscale chamber having a geometry simulating a first in vivo interaction with culture medium, wherein the first chamber comprises a first inlet and a first outlet for flow of the culture medium;

a second microscale chamber having a geometry simulating a second in vivo interaction with the culture medium, wherein the second chamber comprises a second inlet and a second outlet for flow of the culture medium; and

a microfluidic channel interconnecting the first and second chambers.

113. The microscale culture device of claim 112 wherein the chambers are formed from a plastic material.

114. The microscale culture device of claim 113 wherein the plastic material is selected from the group consisting of polystryrene, polymethylmethacrylate, polycarbonate, polytetrafluoroethylene, polyvinylchloride, polydimethylsiloxane, and polysulfone.

115. The microscale culture device of claim 112 and further comprising a pump coupled to the first inlet.

116. The array of microscale culture devices of claim 115 wherein the pump is selected from the group consisting of a peristaltic pump, diaphragm pump, or microelectromechanical pump.

117. The microscale culture device of claim 115 wherein the pump is also coupled to the second outlet to recirculate culture medium.

118. The microscale culture device of claim 112 wherein the chamber geometries are based on a mathematical model representing organs of a living body.

119. The microscale culture device of claim 118 wherein the model is a physiological-based pharmacokinteic model.

120. The microscale culture device of claim 119 wherein the model simulates at least one of known tissue size ratio, tissue to blood volume ratio, and drug residence time.

121. The microscale culture device of claim 112 and further comprising sensors for measuring physiological events in the chambers.

122. The microscale culture device of claim 121 wherein the physiological events comprise cell death, cell proliferation, differentiation, immune response, or perturbations in metabolism or signal transduction pathways.

123. The microscale culture device of claim 121 wherein pharmacokinetic data is derived from the sensors.

124. The microscale culture device of claim 121 wherein the sensors are integrated with the device and provide real-time readout of the physiological status of the cells in the system.

125. A microscale culture device comprising:

a plurality of chambers connected by fluidic passages, each chamber having a geometry simulating parts of a living body; and

a pump, for circulating culture medium through the chambers to simulate the effects of compounds on the living body.

126. The microscale culture device of claim 125 wherein the pump is a peristaltic pump that recirculates the culture medium through the chambers.

127. The microscale culture device of claim 125 wherein the chambers simulate interaction of the culture medium with at least two of a liver, lung, an area of slowly perfused fluid, fat, and an area of rapidly perfused fluid.

128. The microscale culture device of claim 125 wherein one chamber simulates a lung with multiple parallel ridges of material.

129. The microscale culture device of claim 125 wherein one chamber simulates a liver with multiple staggered pillars.

130. The microscale culture device of claim 125 and further comprising a controller.

131. The microscale culture device of claim 130 wherein the controller controls the pump to create culture medium residences times in chambers comparable to those encountered in the living body.

132. The microscale culture device of claim 131 and further comprising valves distributed along the fluid passages, and wherein the controller controls the valves consistent with pharmacokinetic parameter values associated with the simulated parts of the living body.

133. The microscale culture device of claim 130 wherein the chambers are formed on a substrate, and the controller is separate from and electrically coupled to the substrate.

134. The microscale culture device of claim 130 and further comprising a look-up table having pharmacokinetic parameter values associated with the simulated parts of the living body for use by the controller.

135. A microscale culture device, comprising:

a lung simulating chamber;

a pump;

at least two of a liver simulating chamber, a slowly perfused simulating chamber, a rapidly perfused simulating chamber and a fat simulating chamber coupled in parallel; and

a plurality of microfluidic channels serially coupling the lung simulating chamber, the pump, and the at least two chambers.

136. A method of forming a microscale culture device, the method comprising:

forming a first microscale chamber having a geometry simulating a first in vivo interaction with culture medium, wherein the first chamber comprises a first inlet and a first outlet for flow of the culture medium;

forming a second microscale chamber having a geometry simulating a second in vitro interaction with the culture medium, wherein the second chamber comprises a second inlet and a second outlet for flow of the culture medium; and

forming a microfluidic channel interconnecting the first and second chambers.

137. The method of claim 136 wherein the chambers are formed by embossing, injection molding, or stamping.

138. The method of claim 136 and further comprising polymerizing surfaces of the chambers and channel.

139. The method of claim 138 wherein polymeric materials on the surface of the chambers and channels provide enhanced fluid direction, cellular attachment or cellular segregation.

140. An array of microscale culture devices comprising:

a housing for enclosing the devices, each device comprising;

a first microscale chamber having a geometry simulating a first in vivo interaction with culture medium, wherein the first chamber comprises a first inlet and a first outlet for flow of the culture medium; and

channels coupled to the inlets and outlets of the chamber.

141. The array of microscale culture devices of claim 140 wherein each device further comprises a second microscale chamber having a geometry simulating a second in vitro interaction with the culture medium, wherein the second chamber comprises a second inlet and a second outlet for flow of the culture medium with channels coupled thereto.

142. The array of microscale culture devices of claim 140 wherein the devices are coupled in parallel or multiplexed to simulate biological barriers.

143. The array of microscale culture devices of claim 142 wherein the barriers are gastrointestinal barriers or the blood brain barrier.

144. The array of microscale culture devices of claim 140, wherein the number of devices is greater than approximately 10.

145. A system comprising:

a first microscale culture device having

a plurality of microscale chambers with geometries that simulate a plurality of in vivo interactions with a culture medium, wherein each chamber includes an inlet and an outlet for flow of the culture medium, and

a microfluidic channel interconnecting the chambers; and

a control instrument coupled to the first microscale culture device, the control instrument having a computer to acquire data from, and control pharmacokinetic parameters of, the first microscale culture device.

146. The system of claim 145, wherein the first microscale culture device is formed on a computerized chip.

147. The system of claim 145, wherein the first microscale culture device further includes one or more sensors coupled to the control instrument for measuring physiological events in the chambers.

148. The system of claim 147, wherein the sensors include one or more biosensors that monitor the oxygen, carbon dioxide, or pH of the culture medium.

149. The system of claim 145, wherein the control instrument holds the first microscale culture device, and seals a top of the first microscale culture device to establish the microfluidic channel.

150. The system of claim 145, wherein the control instrument further includes

a pump to control circulation of microfluid in the microfluidic channel of the first microscale culture device,

a heating element to control the temperature of the first microscale culture device,

a light source, and

a photodetector to detect fluorescent emissions from cell compartments within the first microscale culture device.

151. The system of claim 150, wherein the computer further records data for fluorescent intensity.

152. The system of claim 151, wherein the computer records data for fluorescent intensity using a measuing instrument of a type that is selected from a group consisting of calorimetric, fluorometric, luminescent, and radiometric.

153. The system of claim 150, wherein the heating element maintains the first microscale culture device at a temperature of 37 degrees Celsius.

154. The system of claim 145, wherein the computer controls a pharmacokinetic parameter selected from a group consisting of group pump speed, temperature, length of experiment, and frequency of data acquisition of the first microscale culture device.

155. The system of claim 145, wherein the computer controls a pharmacokinetic parameter selected from a group consisting of flow rate, chamber geometry, and number of cells in the first microscale culture device.

156. The system of claim 145, wherein the computer further controls one or more pumps in the first microscale culture device to create culture medium residence times in the chambers comparable to those encountered in the living body.

157. The system of claim 145, wherein the computer further controls one or more valves distributed along the microfluidic channel in a manner that is consistent with a pharmacokinetic parameter value associated with a simulated part of a living body.

158. The system of claim 145, and further comprising a second microscale culture device having

a plurality of microscale chambers with geometries that simulate a plurality of in vivo interactions with a culture medium, wherein each chamber includes an inlet and an outlet for flow of the culture medium, and

a microfluidic channel interconnecting the chambers,

wherein the control instrument is coupled to the second microscale culture device.

159. A computerized method for dynamically controlling a microscale culture device, the computerized method comprising:

analyzing data from a plurality of sensors to measure physiological events in a plurality of chambers of the microscale culture device;

regulating fluid flow rates of a culture medium in the chambers of the microscale culture device;

detecting biological or toxicological reactions in the chambers of the microscale culture device; and

upon detection, changing one or more pharmacokinetic parameters of the microscale culture device.

160. The computerized method of claim 159, wherein the detecting includes detecting a change in dimension of a cell compartment of the microscale culture device.

161. The computerized method of claim 159, wherein the changing includes changing a pharmacokinetic parameter selected from a group consisting of interactions between cells, liquid residence time, liquid to cell ratios, metabolism by cells, and shear stress in the microscale culture device.

162. The computerized method of claim 159, wherein the changing includes changing a pharmacokinetic parameter selected from a group consisting of flow rate, chamber geometry, and number of cells in the microscale culture device.

163. The computerized method of claim 159, and further comprising optimizing chamber geometry within the microscale culture device, wherein the optimizing includes

selecting a quantity of chambers,

choosing a chamber geometry that provides a proper tissue or organ size ratio,

choosing an optimal fluid flow rate that provides a proper liquid residence time, and

calculating a cell shear stress.

164. The computerized method of claim 159, and further comprising regulating a temperature of the culture medium.

165. The computerized method of claim 159, and further comprising detecting fluorescent emissions from a cell compartment of the microscale culture device.

166. A computer-readable medium having computer-executable instructions stored thereon to perform a method, the method comprising:

analyzing data from a plurality of sensors to measure physiological events in a plurality of chambers of the microscale culture device;

regulating fluid flow rates of a culture medium in the chambers of the microscale culture device;

detecting biological or toxicological reactions in the chambers of the microscale culture device; and

upon detection, changing one or more pharmacokinetic parameters of the microscale culture device.

167. The computer-readable medium of claim 166, wherein the changing includes changing a pharmacokinetic parameter selected from a group consisting of interactions between cells, liquid residence time, liquid to cell ratios, metabolism by cells, and shear stress in the microscale culture device.

168. The computer-readable medium of claim 166, wherein the changing includes changing a pharmacokinetic parameter selected from a group consisting of flow rate, chamber geometry, and number of cells in the microscale culture device.

169. The computer-readable medium of claim 166, wherein the method further comprises optimizing chamber geometry within the microscale culture device, and wherein the optimizing includes

selecting a quantity of chambers,

choosing a chamber geometry that provides a proper tissue or organ size ratio,

choosing an optimal fluid flow rate that provides a proper liquid residence time, and

calculating a cell shear stress.

170. The computer-readable medium of claim 166, wherein the method further comprises regulating a temperature of the culture medium.

171. The computer-readable medium of claim 166, wherein the method further comprises detecting fluorescent emissions from a cell compartment of the microscale culture device.

172. A computer comprising:

a microprocessor;

a general memory;

a non-volatile storage element;

an input/output interface that includes an interface to a microscale culture device having one or more sensors; and

computer software executable on the microprocessor to

analyze data from the sensors to measure physiological events in a plurality of chambers of the microscale culture device,

regulate fluid flow rates of a culture medium in the chambers of the microscale culture device,

detect biological or toxicological reactions in the chambers of the microscale culture device, and

upon detection, change one or more pharmacokinetic parameters of the microscale culture device.

173. The computer of claim 172, wherein the non-volatile storage element includes historical data taken from published information, data gathered from previously run tests, or data derived from theoretical calculations.

174. The computer of claim 172, wherein the computer software regulates the fluid flow rates by transmitting commands to one or more pumps of the microscale culture device through pump control lines.

175. The computer of claim 172, wherein the computer software is further executable on the microprocessor to regulate a temperature of the culture medium.

176. The computer of claim 175, wherein the computer software regulates the temperature by transmitting commands to a heater coil of the microscale culture device through heater coil control lines.

177. The computer of claim 172, and further comprising:

a look-up table memory coupled to the general memory for storing a set of mass balance equations that represent physiologically-based pharmacokinetic models for various biological or chemical substances in the system; and

a cache memory coupled to the microprocessor for storing the computer software.

178. The computer of claim 172, wherein the input/output interface further includes a keyboard interface,

a display interface, and

a printer/plotter recorder interface.