Reversible stencils for fabricating micro-tissues

Patent metadata

Field	Value
Publication number	US-10851344-B2
Application number	US-201515329035-A
Country	US
Kind code	B2
Filing date	Aug 7, 2015
Priority date	Aug 7, 2014
Publication date	Dec 1, 2020
Grant date	Dec 1, 2020

Abstract

The invention relates to devices, methods, kits, and compositions for in vitro generation of three-dimensional micro-tissues that are accurate models of heart, skeletal muscle, neuronal, and other tissues.

First claim

What is claimed:

1. A method of inducing self-assembly of mammalian cells into one or more dog-bone shaped micro-tissues comprising
seeding the mammalian cells into one or more microwells of a device comprising
a cell adhesion substrate; and
a removable elastomeric stencil overlay comprising one or more cut-out patterned microwells comprising two or more circular, oval, rectangular, square, V-shaped, or triangular holes, each hole joined to an adjacent hole by a canal; and

culturing the seeded cells within the microwells, to thereby induce self-assembly of the mammalian cells into one or more dog-bone micro-tissues shaped within adjacent holes and along the canal joining the adjacent holes;
wherein the cell adhesion substrate binds cells within at least the holes of the cut-out pattern.

2. The method of claim 1 , wherein about 2000 to about 9500 cells are seeded into each of several microwells of the device.

3. The method of claim 1 , wherein each of the microwells has a depth of at least 250 μm or at least 500 μm.

4. The method of claim 1 , wherein at least one of the microwells has at least two square holes with a side length “L” of 250 μm to 1000 μm, and the canal joining the two square holes has a longitudinal length “X” of 250-1000 μm a transverse width “Y” of 50-200μm.

5. The method of claim 1 , wherein the ratio of L to Y is at least five.

6. The method of claim 1 , wherein the seeded cells are a mixture of mammalian cell types typically present in a mammalian organ.

7. The method of claim 6 , wherein the mammalian organ is selected from the group consisting of heart, muscle, and neuronal tissue.

8. The method of claim 1 , wherein the mammalian cells comprise a heterologous marker gene, a heterologous reporter gene, a mutant gene, or a combination thereof.

9. The method of claim 1 , wherein the mammalian cells comprise a marker gene encodes a fluorescent protein.

10. The method of claim 1 , wherein the mammalian cells comprise a mutation in any of the following genes: ABCC9, ACTC1, ACTN2, ANKRD1, AKAP9, ANK2, BAG3, CACNA1C, CACNB2, CASQ2, CAV3, COX15, CRYAB, CSRP3, CTF1, DES, DMD, DNAJC19, DSC2, DSG2, DSP, DTNA, EYA4, FHL2, FKTN, FOXD4, GLA, KCNE1, KCNE2, KCNH2, KCNJ5, KCNJ8, KCNQ1, KCNQ2, LAMA4, LAMP2, LDB3, LMNA, MYBPC3, MYH6, MYH7, MYL2, MYL3, MYOZ2, NEXN, PKP2, PLN, PRKAG2, PSEN1, PSEN2, RBM20, RYR2, SCN5A, SDHA, SGCD, SNTA1, SYNE1, SYNE2, TAZ, TCAP, TMEM43, TMPO, TNNC1, TNNT2, TNNC1, TNNI3, TPM1, TRDN, TTN, TTR, VCL, or any combination thereof.

11. The method of claim 1 , wherein seeding the mammalian cells comprises settling the cells into the microwells by gravity or by fluid flow through a membrane at the bottom of the microwells.

12. The method of claim 1 , further comprising introducing a test compound, oligonucleotide, nucleic acid, protein, or a combination thereof into one or more microwells while culturing the seeded cells within the microwells.

13. The method of claim 1 , further comprising introducing a test compound, oligonucleotide, nucleic acid, protein, or a combination thereof into one or more microwells via membrane at the bottom of the microwells, where the microwells are operably connected to a network of microfluidic channels for introduction of the test compound, oligonucleotide, nucleic acid, protein, or a combination thereof.

14. The method of claim 1 , further comprising determining whether cells are aligned in one or more canals of one or more of the microwells.

15. The method of claim 1 , further comprising determining whether cells have formed dog-bone shaped structures in one or more canals or holes of the microwells.

16. The method of claim 1 , further comprising determining whether cells are contracting along the longitudinal axis of one or more of the microwells.

17. The method of claim 1 , further comprising culturing one or more micro-tissues in a medium containing a test agent.

18. The method of claim 1 , further comprising determining micro-tissue morphology, genetic expression, contraction rate, contraction intensity, electrical activity, calcium transient amplitude, intracellular Ca 2+ level, cell size contractile force production, sarcomeric α-actinin distribution, or a combination thereof.

19. The method of claim 1 , wherein cells within the micro-tissues formed in the microwells exhibit contractility with greater synchronicity than two-dimensional monolayers of the same cell type and composition.

20. The method of claim 1 , wherein cells within the micro-tissues formed in the microwells respond to drugs with greater synchronicity than two-dimensional monolayers of the same cell type and composition.

21. The method of claim 1 , wherein cells within the micro-tissues formed in the microwells exhibit more synchronized chronotropic and/or inotropic responses to drugs compared to than two-dimensional monolayers of the same cell type and composition.

22. The method of claim 1 , further comprising removing the stencil to generate intact micro-tissues.

23. The method of claim 1 , further comprising recovering cells from the microwells and determining expression of one or more mRNA or protein.

24. The method of claim 1 , further comprising immersing one or more micro-tissues in a support medium, damaging one or more micro-tissues, embedding one or more micro-tissues, fixing one or more micro-tissues, fixing one or more micro-tissues, freezing one or more micro-tissues, sectioning one or more micro-tissues, staining one or more micro-tissues, or a combination thereof.

25. The method of claim 1 , wherein the mammalian cells seeded in the microwells are wild type or mutant somatic cells converted into induced pluripotent stem cells and then differentiated into a desired lineage.

Assignees

• Univ California
• The J David Gladstone Inst A Testamentary Trust Established Under The Will Of J David Gladstone

Inventors

• Huebsch Nathaniel
• Conklin Bruce
• Healy Kevin E
• Loskill Peter

Classifications

• C12N2535/10

  Patterned coating · CPC title

• C12N5/0657 Primary

  Cardiomyocytes; Heart cells · CPC title

• C12N2533/30

  Synthetic polymers · CPC title

• C12M25/14

  Scaffolds; Matrices (in general C12N5/0068) · CPC title

• C12M21/08

  for producing artificial tissue or for ex-vivo cultivation of tissue (prostheses A61F2/00, grafts A61L27/00) · CPC title