Plant-derived nanofibrillar cellulose hydrogel for cell culture and chemical testing

Patent metadata

Field	Value
Publication number	US-10976308-B2
Application number	US-201515536558-A
Country	US
Kind code	B2
Filing date	Dec 18, 2015
Priority date	Dec 18, 2014
Publication date	Apr 13, 2021
Grant date	Apr 13, 2021

Abstract

The present invention relates a method for chemical testing, comprising culturing cells in a first plant-derived nanofibrillar cellulose (NFC) hydrogel to obtain in vivo like cells; exposing the in vivo like cells to a test chemical; optionally within another plant-derived NFC hydrogel; incubating the exposed in vivo like cells; detecting during or after incubating, the impact of the test chemical on the in vivo like cells by at least one detection; and removing the plant-derived NFC hydrogel at least once at any stage after obtaining the in vivo like cells and before at least one detection used for detecting the impact of the test chemical on the in vivo like cells. The invention further relates to the use of plant-derived NFC hydrogel in a method for chemical testing, the use of in vivo like cells obtained by culturing cells in plant-derived NFC hydrogel for chemical testing and to a kit for chemical testing comprising plant-derived NFC hydrogel, instructions and a cell or test chemical library.

First claim

The invention claimed is:

1. A method for culturing and chemical testing of in vivo like cells, the method comprising:
a) culturing cells on or in a first plant-derived nanofibrillar cellulose (NFC) hydrogel to obtain in vivo like cells;
b) exposing the in vivo like cells to a test chemical within a second plant-derived NFC hydrogel after removing at least some of the first NFC hydrogel, the first and second NFC hydrogel having a number average diameter of about 1-20 nm, the second hydrogel having a different NFC concentration than the first NFC hydrogel;
c) incubating the exposed in vivo like cells; and
d) detecting, during or after the incubation, a qualitative and/or quantitative impact of the test chemical on the in vivo like cells, by at least one detection technique, the at least one detection technique selected from the group consisting of chromatography, spectroscopy, microscopy, photometry, laser or flow-cytometry, high-throughput screening, or any combination thereof.

2. The method according to claim 1 , wherein the removing the at least some of the first plant-derived NFC hydrogel comprises physical removing, mechanical removing, chemical removing, or any combination thereof.

3. The method according to claim 1 , further comprising removing the second plant-derived NFC hydrogel between b) and d).

4. The method of claim 1 , further comprising:
e) removing the second NFC hydrogel to separate the second NFC hydrogel from the in vivo like cells at any stage after b) and before the at least one detection technique according to d).

5. The method of claim 4 , wherein, during e), the second plant-derived NFC hydrogel is treated enzymatically with a cellulase for a time sufficient to at least partly release the cells.

6. The method according to claim 5 , wherein the cellulase is a cellulolytic enzyme mixture.

7. The method of claim 6 , wherein the cellulase comprises hemicellulases, a commercial cellulase, a partially purified cellulase, or a purified cellulase.

8. The method according to claim 1 , wherein the second hydrogel has different properties than the first NFC hydrogel, the properties selected from the group consisting of one or more of size distribution, length, diameter, chemical composition, or rheological properties.

9. The method according to claim 1 , wherein the second NFC hydrogel has different properties than the first NFC hydrogel, the different properties being selected from the group consisting of stiffness of NFC hydrogel, shear-zero viscosity of NFC hydrogel, charge of NFC, and transparency of NFC hydrogel.

10. The method according to claim 1 , wherein said chemical testing comprises toxicity testing; safety testing; drug candidate testing; drug screening; or pro-drug candidate testing.

11. The method of claim 1 , wherein the chemical testing is for genotoxicity, carcinogenicity, neurotoxicity, mitochondrial toxicity, cardiotoxicity, hepatotoxicity, hematopoietic toxicity, nephrotoxicity, safety testing, causing reproductive toxicity, skin irritation, eye irritation, or any combination thereof.

12. The method according claim 1 , wherein said test chemical is selected from the group consisting of drugs, drug candidates, pro-drugs, pro-drug candidates, nanoparticles, cell regulatory agents, food or food additives, household products, industrial chemicals, packing materials, air freshener, plant growth regulatory agents, environmental toxins, pesticides, personal care products, or their chemical ingredients.

13. The method according to claim 1 , wherein said in vivo like cells are stem cells, primary cells, secondary cells, or any combinations thereof.

14. The method according to claim 1 , wherein the first NFC hydrogel is provided onto a support before cells for culturing are seeded onto or into the first NFC hydrogel or after cells for culturing are seeded into the first NFC hydrogel.

15. The method according to claim 1 , wherein the detection of the impact of the test chemical on the in vivo like cells further comprises transplantation of the exposed in vivo like cells into test animals.

16. The method of claim 1 , wherein, during the incubating, the exposed in vivo like cells absorb, bind to, or otherwise react with the test chemical.

17. The method according to claim 1 , wherein said first or second NFC hydrogel includes from 0.05 to 10 wt % of nanofibrillar cellulose.

18. The method according to claim 1 , wherein said first or second NFC hydrogel comprises native nanofibrillar cellulose.

19. The method according to claim 1 , wherein said first or second NFC hydrogel comprises nanofibrillar cellulose including cellulose I.

20. The method according to claim 1 , wherein said first or second NFC hydrogel has a stiffness of at least 4 Pa.

21. The method of claim 1 , wherein at least one of the first or second NFC hydrogel is anionic.

22. The method of claim 1 , wherein at least one of the first or second NFC hydrogels has a turbidity of about 80 NTU or less.

23. The method according to claim 1 , wherein the detection technique is chromatography, the chromatography selected from the group consisting of Gas Chromatography (GC), High Performance Liquid Chromatography (HPLC), affinity chromatography, displacement chromatography, ion-exchange chromatography, size exclusion chromatography, gel-filtration chromatography, fast protein liquid chromatography, paper chromatography, thin-layer chromatography, electrochromatography, or any combination thereof.

24. The method of claim 23 , wherein the electrochromatography is selected from the group consisting of gel-electrophoresis, 2D gel-electrophoresis, isoelectric focusing, or any combination thereof.

25. The method of claim 1 , wherein the detection technique is spectroscopy, the spectroscopy selected from the group consisting of nuclear magnetic resonance (NMR) spectroscopy, Raman spectroscopy, Infrared spectroscopy (IR), Ultraviolet spectroscopy (UV), visible light spectroscopy, fluorescence spectroscopy, mass spectrometry (MS), or any combination thereof.

26. The method of claim 1 , wherein the detection technique is microscopy, the microscopy selected from the group consisting of optical microscopy, electron microscopy, scanning-probe microscopy, or any combination thereof.

27. The method of claim 26 , wherein the optical microscopy is selected from the group consisting of phase contrast microscopy, reverse phase contrast microscopy, confocal microscopy, fluorescence microscopy, or any combination thereof.

28. The method of claim 26 , wherein the electron microscopy is selected from the group consisting of Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), or any combination thereof.

29. The method of claim 26 , wherein the scanning probe microscopy is Atomic Force Microscopy (AFM).

Assignees

• Upm Kymmene Corp

Inventors

• Yliperttula Marjo
• Urtti Arto
• Lou Yan-Ru
• Malinen Melina
• Laurén Patrick
• Kanninen Liisa
• Niklander Kirsti Johanna
• Somersalo Petter
• Harjumäki Riina

Classifications

• C12N5/00

  Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; (plant reproduction by tissue culture techniques A01H4/00) · CPC title

• C12N2539/10

  Coating allowing for selective detachment of cells, e.g. thermoreactive coating · CPC title

• G01N33/5082 Primary

  Supracellular entities, e.g. tissue, organisms · CPC title

• G01N33/5436

  with ligand physically entrapped within the solid phase (liposomes G01N33/5432; immunological test elements G01N33/54386) · CPC title

• C12N5/0606

  Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696) · CPC title