Vascularized perfused microtissue/micro-organ arrays

• US 6,197,575 B1
• Filed: 03/18/1999
• Issued: 03/06/2001
• Est. Priority Date: 03/18/1998
• Status: Expired due to Term

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Thompson, Charles D., Domansky, Karel, Powers, Mark J., Tannenbaum, Steven, Griffith, Linda G.
• Primary Examiner(s): Beisner, William H.
• Application Number:
  US09/272,227
• Time in Patent Office: 719 Days
• Field of Search: 435/1.1, 435/1.2, 435/29, 435/32, 435/33, 435/284.1, 435/287.1, 435/395, 435/401, 435/288.3, 435/402, 435/288.4, 435/373, 435/288.5, 435/288.7, 435/297.2, 435/297.5, 435/305.1, 435/305.2, 435/DIG.6, 435/DIG-43-, 435/DIG.47
• US Class Current: 435/288.4
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00308 interchangeably mounted in racks or blocks
• CPC Class Codes: B01J 2219/00313 the reactor vessels being formed by arrays of wells in blocks
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00319 the blocks being mounted in stacked arrangements
• CPC Class Codes: B01J 2219/00351 Means for dispensing and evacuation of reagents
• CPC Class Codes: B01J 2219/00376 in multiple or parallel arrangements
• CPC Class Codes: B01J 2219/00416 Vacuum
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/0072 Organic compounds
• CPC Class Codes: B33Y 10/00 Processes of additive manufacturing
• CPC Class Codes: B33Y 80/00 Products made by additive manufacturing
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C09K 8/685 containing cross-linking agents
• 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 23/12 Well or multiwell plates C12M25/04 takes precedence
• CPC Class Codes: C12M 41/46 of cellular or enzymatic activity or functionality, e.g. cell viability
• CPC Class Codes: C40B 60/14 for creating libraries
• 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 33/502 : for testing non-proliferative effects
• CPC Class Codes: G01N 33/5038 : involving detection of metabolites per se
• CPC Class Codes: G01N 33/5058 : Neurological cells
• CPC Class Codes: G01N 33/5064 : Endothelial cells
• CPC Class Codes: G01N 33/5073 : Stem cells

Abstract

Systems including (1) a micromatrix and perfusion assembly suitable for seeding and attachment of cells within the matrix and for morphogenesis of seeded cells into complex, hierarchical tissue or organ structures, wherein the matrix includes channels or vessels through which culture medium, oxygen, or other nutrient or body fluids can be perfused while controlling gradients of nutrients and exogenous metabolites throughout the perfusion path independently of perfusion rate, and (2) sensor means for detecting changes in either cells within the matrix or in materials exposed to the cells, have been developed. Methods for making the micromatrices include micromachining, micromolding, embossing, laser drilling, and electro deposition machining. Cells can be of one or more types, either differentiated or undifferentiated. In a preferred embodiment, the matrix is seeded with a mixture of cells including endothelial cells which will line the channels to form “blood vessels”, and at least one type of parenchymal cells, such as hepatocytes, pancreatic cells, or other organ cells. The system can be used to screen materials for an effect on the cells, for an effect of the cells on the materials (for example, in a manner equivalent to tissue metabolism of a drug), or to test a material on a biological that must first infect cells or tissues, such as viruses. The apparatus also can be used to provide a physiological environment for expansion of stem cells, or for enabling gene therapy in vitro.

Claims

1. An apparatus comprising

a) a matrix comprising one or more channels which support the viability of cells,

b) endothelial cells within the channels of the matrix, and

c) means for detecting changes in the cells or in compounds exposed to the cells,

wherein the matrix allows perfusion of the cells with nutrients and oxygen sufficient to maintain the viability of the cells and the matrix allows formation of microvascular networks, wherein the nutrients are provided by culture medium which perfuses through the microvascular networks.

2. The apparatus of claim 1 wherein the matrix comprises a silicon chip with an array of open channels.

3. The apparatus of claim 2 wherein the channels have a depth of between 150 and 400 micrometers.

4. The apparatus of claim 1 wherein means for detecting changes in the cells or in compounds exposed to the cells comprises a miniaturized fiber optic system which provides single or multi-photon excitation to individual channels.

5. The apparatus of claim 1 wherein the channels are coated with a substance selected from the group consisting of peptides or proteins promoting cell adhesion on biocompatible polymers.

6. The apparatus of claim 1 wherein the matrix comprises a polymer with an array of open channels.

7. The apparatus of claim 1 wherein the matrix is prepared using a method selected from the group consisting of micromolding, electro-deposition machining, laser ablation, laser drilling, micromachining, wet etching, reactive ion etching, and embossing.

8. The apparatus of claim 1 wherein the matrix further comprises a bioactive agent.

9. The apparatus of claim 8 wherein the bioactive agent is selected from the group consisting of cell growth, differentiation, and migration modulators.

10. The apparatus of claim 1 wherein the means for detecting changes in the cells or in compounds exposed to the cells comprises sensors which detect changes in pH, oxygen levels, specific metabolites, or the presence or absence of an indicator molecule.

11. The apparatus of claim 1 further comprising cells selected from the group consisting of parenchymal cells which provide metabolic or organ function, nerve or nervous tissue, connective tissue, and undifferentiated cells, wherein the cells are attached to the endothelial cells within the channels of the matrix.

12. The apparatus of claim 11 wherein the undifferentiated cells are selected from the group consisting of embryonic cells, stem cells, and other precursor cells.

13. The apparatus of claim 1 wherein the cells are transgenic for one or more genes.

14. The apparatus of claim 1 wherein the surface of the channel walls is grooved or scalloped for controlling cell adhesion, spreading, morphogenesis, or function.

15. The apparatus of claim 1 which is suitable for cryopreservation of tissue structures.

16. The apparatus of claim 1 wherein the rate of cell perfusion is controllable via a flow of perfusate.

17. The apparatus of claim 1 wherein the flow is across at least one side of the channels or is forced through the channels.

18. The apparatus of claim 1 wherein the matrix comprises two or more independent perfusion circuits.

19. The apparatus of claim 18 wherein one circuit is above the channel and one circuit is below the channel.

20. The apparatus of claim 1 wherein the cells are attached within the channel.

21. The apparatus of claim 1 wherein the cells form aggregates or spheroids within the channel.

22. A method for screening drugs for biological activity, toxicity or teratogenicity comprising adding a drug to an apparatus which comprises:

a) a matrix comprising one or more channels which support the viability of cells,

b) cells forming microvascular networks within the channels of the matrix, and

c) means for detecting changes in the cells or in compounds exposed to the cells,

wherein the cells are perfused with nutrients and oxygen through the microvascular network sufficient to maintain the viability of the cells.

23. A method for propagating stem cells comprising:

a) providing an apparatus which comprises;

a matrix, which includes;

i) one or more channels within the matrix,

ii) endothelial cells within the channels of the matrix, and

iii) means for detecting changes in the cells or in compounds exposed to the cells,

wherein the matrix allows perfusion through the channels of the matrix of the cells with nutrients and oxygen sufficient to maintain the viability of the cells; and

b) propagating the cells on the matrix.

24. The method of claim 23 further comprising adding a soluble compound to the medium which causes the stem cells to extravasate from the matrix.

25. The method of claim 24 wherein the compound is a chemotherapeutic agent.

26. The method of claim 24 further comprising harvesting the cells.

27. The method of claim 26 wherein the cells are harvested by collection on a filter.

28. A method for screening a compound for at least one activity under physiological conditions in a microarray comprising exposing cells in an apparatus, which comprises a matrix, which includes:

i) one or more channels within the matrix,

ii) endothelial cells within the channels of the matrix, and

iii) means for detecting changes in the cells or in compounds exposed to the cells,

wherein the cells form microvascular networks in the channels of the matrix and the channels allow perfusion of the cells with nutrients and oxygen sufficient to maintain the viability of the cells exposed to a compound to be tested and screened for at least one effect of the compound on the cells.