Cell harvest method

Patent metadata

Field	Value
Publication number	US-2023190820-A1
Application number	US-202117927415-A
Country	US
Kind code	A1
Filing date	May 27, 2021
Priority date	May 27, 2020
Publication date	Jun 22, 2023
Grant date	—

Abstract

The invention generally relates to cells and compositions comprising same for use in cell therapy, to methods of obtaining same, and to use of same in cell therapy. In one aspect, the invention provides a method for forming a cell composition from a tissue sample, the method comprising: providing a tissue sample comprising cells; contacting the sample with a polymer in binding conditions, said binding conditions being conditions that enable binding of cells in the sample to the polymer, so that said cells are bound to the polymer; culturing the cells bound to the polymer under conditions and for a time that allows the cell number to increase; providing conditions to induce a phase change of the polymer; thereby forming a cell composition from a tissue sample.

First claim

1 . A method for forming a cell composition from a tissue sample, the method comprising:
- providing a tissue sample comprising cells; - contacting the sample with a polymer in binding conditions, said binding conditions being conditions that enable binding of cells in the sample to the polymer, so that said cells are bound to the polymer; - culturing the cells bound to the polymer under conditions and for a time that allows the cell number to increase; - providing conditions to induce a phase change of the polymer, wherein the cells remain bound to the phased changed polymer; thereby forming a cell composition from a tissue sample.

2 . The method of claim 1 , further comprising a step of isolating the cells from the extracellular matrix in the tissue sample.

3 . The method of claim 2 , wherein isolating the cells from the extracellular matrix is performed by mechanical disruption.

4 . The method of claim 2 , wherein isolating the cells from the extracellular matrix is performed by enzymatic digestion.

5 . The method of any one of claims 2 to 4 , wherein isolating the cells from the extracellular matrix is performed by mechanical disruption and enzymatic digestion.

6 . The method of any one of claims 2 to 5 , wherein isolating the cells separates the cells from any fat lobules in the sample.

7 . The method of claim 4 or 5 , wherein the enzymatic digestion is performed with collagenase.

8 . The method of claim 7 , wherein the collagenase is used at a specific activity of 2 U/ml for a period of 30 minutes or less.

9 . The method of any one of claims 2 to 8 , further comprising the step of separating the isolated cells from substantially all the fat and/or liquid present in the sample.

10 . The method of claim 9 , wherein separating the isolated cells may be performed by centrifugation.

11 . The method of claim 10 , wherein the centrifugation is performed at about 2000 g for about 5 minutes to form a cell pellet.

12 . The method of claim 11 , wherein the cell pellet is resuspended in a buffer for lysis of red blood cells.

13 . The method of claim 12 , further comprising filtering the cells in the lysis buffer to separate debris from viable cells and further centrifugation for about 5 minutes at about 400 g to obtain a further cell pellet.

14 . The method of claim 1 , wherein the polymer is capable of attaching to a solid phase of a particle, vessel or device, or capable of forming a particle, in said binding conditions.

15 . The method of any one of claims 1 to 14 , wherein the polymer is capable of binding to cells in said binding conditions that are human adipose derived stem cells (ADSCs) or hADSC precursor cells, or to cells that are derived from hADSC that are chondrogenic or that have chondrogenic potential.

16 . The method of any one of claims 1 to 15 , wherein the polymer is not capable of binding to fibroblasts in said binding conditions.

17 . The method of any one of the claims 1 to 16 , wherein the polymer comprises a peptide or protein.

18 . The method of claim 17 , wherein the peptide or protein binds to an extracellular matrix adhesion receptor.

19 . The method of claim 18 , wherein the extracellular matrix adhesion receptor is an integrin receptor.

20 . The method of claim 17 , wherein the peptide or protein comprises an integrin binding motif.

21 . The method of claim 20 , wherein the integrin binding motif is RGD.

22 . The method of claim 17 , wherein the peptide comprises or consists of GGGGRGDSP, GRGDSP or GRGDS, or an amino acid sequence with 1 or 2 amino acid insertions, deletions, substitutions (preferably conservative substitutions) or a combination thereof.

23 . The method of any one of claims 1 to 22 , wherein the polymer is capable of reversible liquid-solid phase change.

24 . The method of any one of claims 1 to 23 , wherein the polymer is capable of a liquid to solid phase change caused by an ionic crosslinking agent.

25 . The method of claim 24 , wherein the ionic crosslinking agent is a divalent cation.

26 . The method of claim 25 , wherein divalent cation is Ca 2+ .

27 . The method of any one of claims 1 to 26 , wherein the polymer is capable of a solid to liquid phase change caused by the chelation of an ionic crosslinking agent.

28 . The method of claim 27 , wherein the chelation occurs by presence of a chelating agent capable of chelating an ionic crosslinking agent.

29 . The method of claim 26 , wherein the chelating agent is EDTA.

30 . The method of any one of the claims 1 to 29 , wherein the polymer comprises gelatin or a derivative thereof.

31 . The method of claim 30 , wherein the gelatin polymer is Gelatin methacryloyl (GeIMA).

32 . The method of any one of claims 1 to 29 , wherein the polymer comprises alginate or derivative thereof.

33 . The method according to claim 32 , wherein the polymer comprises alginate-RGD.

34 . The method according to any one of claims 1 to 33 , wherein the polymer is capable of photo-crosslinking.

35 . The method according to claim 34 , wherein the photo-crosslinking is mediated by a reactive functionality capable of photo-crosslinking present in the polymer.

36 . The method according to claim 35 , wherein the reactive functionality is a methacryloyl group.

37 . The method of any one of claims 1 to 36 , wherein the polymer comprises alginate, an RGD motif and a methacryloyl group.

38 . The method of any one of claims 1 to 37 , wherein the tissue sample comprises a first cell type and a second cell type and wherein the binding conditions enable the binding of the first cell type to the polymer and wherein the binding conditions do not enable binding of the second cell type to the polymer.

39 . The method of claim 38 , wherein separation of the polymer from the tissue sample forms a cell composition consisting of cells of the first cell type, and forms a waste stream comprising cells of the second cell type.

40 . The method of claims 38 or 39 , wherein the first cell type is a hADSC or chondrogenic cell and the second cell type is a fibroblast.

41 . The method of any one of claims 1 to 40 , wherein the tissue sample is obtained from the infrapatellar fat pad.

42 . The method of claim 41 , wherein the fat pad has a weight of about 2 to 3 g.

43 . The method of any one of claims 1 to 42 , wherein the polymer is in the form of a 3D particle.

44 . The method of claim 43 , wherein the sample is contacted with the 3D particle in a bioreactor at a cell:particle ratio of about 10 cells to every particle.

45 . The method of any one of claims 1 to 44 , wherein the step of culturing the cells allows at least 2 cycles of cell divisions.

46 . The method of any one of claims 1 to 44 , wherein the step of culturing the cells is for a period of at least 5, at least 6 or at least 7 days.

47 . The method of any one of claims 1 to 44 , wherein the step of culturing the cells results in an increase of about 3-4 times the original cell number.

Assignees

• Univ Melbourne
• Univ Swinburne Technology
• Univ Wollongong
• St Vincents Hospital Melbourne Ltd

Inventors

• Choong Peter F M
• Francis Sam Lourdesan
• Duchi Serena
• Onofrillo Carmine
• Bella Claudia Di
• Gambhir Sanjeev
• Moulton Simon Edward
• Halkias Christopher
• O'Connell Cathal D
• Reynolds Nicholas Paul
• Wallace Gordon George

Classifications

• A61K47/183

  Amino acids, e.g. glycine, EDTA or aspartame · CPC title

• A61K41/00

  Medicinal preparations obtained by treating materials with wave energy or particle radiation {; Therapies using these preparations} · CPC title

• C12N2509/00

  Methods for the dissociation of cells, e.g. specific use of enzymes · CPC title

• A61K47/42

  Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein (oligopeptides having up to five amino acids {A61K47/183}; polyamino acids A61K47/34) · CPC title

• C12N2537/10

  Cross-linking · CPC title