Composition and methods for culturing cells

Patent metadata

Field	Value
Publication number	US-2016201031-A1
Application number	US-201414912384-A
Country	US
Kind code	A1
Filing date	Aug 20, 2014
Priority date	Aug 21, 2013
Publication date	Jul 14, 2016
Grant date	—

Abstract

The present disclosure provides compositions for in vitro culture of cells. The present disclosure provides methods for in vitro culture of cells. The present disclosure provides methods for extracting proteins from a serum sample.

First claim

What is claimed is:

1 . A method for increasing the metabolic activity of a non-phagocytic cell, the method comprising:
culturing the non-phagocytic cell in a culture composition comprising a population of nanoparticles of poly(ethyl acrylate-co-methyl methacrylate-co-trimethylammonioethyl methacrylate chloride) 1:2:0.1 for a period of time sufficient to increase the metabolic activity of the non-phagocytic cell, wherein the average diameter of the nanoparticles is from 30 nm to 100 nm.

2 . The method according to claim 1 , wherein the culture composition further comprises a growth factor.

3 . The method according to claim 1 or claim 2 , wherein the culture composition further comprises animal serum.

4 . The method according to claim 3 , wherein the animal serum is fetal bovine serum.

5 . The method according to any of claims 1 to 4 , wherein the culture composition comprises conditioned media.

6 . The method according to any of claims 1 to 5 , wherein the cell is a primary cell.

7 . The method according to claim 6 , wherein the primary cell is selected from: a primary epithelial cell, and a cancer cell.

8 . The method according to claim 7 , wherein the primary epithelial cell is a primary human breast cell.

9 . The method according to any of claims 1 to 8 , wherein the cell is a cancer cell.

10 . The method according to claim 9 , wherein the cancer cell is a breast cancer cell.

11 . The method according to any of claims 1 to 10 , wherein the cell is a neural progenitor cell.

12 . The method according to any of claims 1 to 11 , wherein the average diameter of the nanoparticles of the population is from 55 nm to 90 nm.

13 . The method according to any of claims 1 to 11 , wherein the average diameter of the nanoparticles of the population is from 60 nm to 80 nm.

14 . The method according to any of claims 1 to 13 , wherein metabolic activity of the cell is increased relative to a control cell.

15 . A method for increasing the metabolic activity of a non-phagocytic cell, the method comprising:
(a) contacting the cell with a culture medium so that the culture medium comprises the cell; and (b) contacting the culture medium comprising the cell with a population of at least two nanoparticles of poly(ethyl acrylate-co-methyl methacrylate-co-trimethylammonioethyl methacrylate chloride) 1:2:0.1 for a period of time sufficient to increase the metabolic activity of the non-phagocytic cell, wherein, the average diameter of the nanoparticles of the population is from 55 nm to 100 nm.

16 . The method according to claim 15 , wherein the culture medium comprises a growth factor.

17 . The method according to claim 15 or 16 , wherein the culture medium comprises animal serum.

18 . The method according to any of claims 15 to 17 , wherein the cell is not contacted with a serum-free culture medium prior to step (b).

19 . The method according to any of claims 15 to 18 , wherein the population of at least two nanoparticles is a population of at least two naked nanoparticles.

20 . The method according to any of claims 15 to 19 , wherein the contacting of step (b) is for a period of time greater than 48 hours.

21 . The method according to any of claims 15 to 19 , wherein the contacting of step (b) is for a period of from 24 hours to 72 hours.

22 . The method according to any of claims 15 to 21 , further comprising measuring the metabolic activity of the non-phagocytic cell.

23 . The method according to claim 22 , wherein measuring the metabolic activity of the cell is performed using a tetrazolium dye other than MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole).

24 . A method of extracting proteins from a serum sample, the method comprising:
(a) contacting a serum sample with a population of nanoparticles of poly(ethyl acrylateco-methyl methacrylate-co-trimethylammonioethyl methacrylate chloride) 1 : 2 : 0 . 1 for a period of time sufficient for the nanoparticles to bind to proteins present in the serum, to obtain a population of protein-bound nanoparticles; and (b) isolating protein-bound nanoparticles of the population of protein-bound nanoparticles, to obtain a population of isolated protein-bound nanoparticles; thereby extracting proteins from the serum sample.

25 . The method according to claim 24 , further comprising contacting a cell with isolated protein-bound nanoparticles of the population of isolated protein-bound nanoparticles.

26 . The method according to claim 24 or 25 , further comprising identifying a protein, or an Interpro domain of a protein, bound to an isolated protein-bound nanoparticle of the population of isolated protein-bound nanoparticles.

27 . The method of claim 26 , where identifying comprises the using mass spectrometry.

28 . The method according to claim 26 , comprising identifying an Interpro domain of a protein bound to an isolated protein-bound nanoparticle of the population of isolated protein-bound nanoparticles.

29 . The method according to claim 28 , wherein the identified Interpro domain is selected from: IPR006210:EGF-like, IPR000215:Protease_inhib_I4_serpin, IPR013783:Ig-like_fold, IPR016060:Complement_control_module, IPRO01254:Peptidase_S1_S6, IPRO01611:Leu-rich_rpt, IPR006209:EGF, IPR008985:ConA-likelec_gl, IPR000436:Sushi_SCR_CCP, IPR009003:Pept_cys/ser_Trypsin-like, IPR018039:Intermediate_filament_CS, IPRO11992:EF-hand-like_dom, PR002035:VWF_A, IPR001599:Macroglobln_a2, IPR000859:CUB, IPR008160:Collagen, IPR003961:Fibronectin_type3, IPRO04001:Actin_CS, IPR000264:Serumumin, IPR000001:Kringle, IPRO17857:Coagulation_fac_subgr_Gla_dom, IPR009053:Prefoldin, IPR018056:Kringle_CS, IPR000719:Prot_kinase_cat_dom, IPRO01791:Laminin_G, IPR000010:Prot_inh_cystat, IPR000884:Thrombospondin_1_rpt, IPRO12674:Calycin, IPR008979:Galactose-bd-like, IPR020837:Fibrinogen_CS, IPRO01304:C-typelectin, IPR002223:Prot_inh_Kunz-m, and IPRO17441:Protein_kinase_ATP_BS.

30 . The method according to claim 26 , comprising identifying a protein bound to an isolated protein-bound nanoparticle of the population of isolated protein-bound nanoparticles.

31 . The method according to claim 30 , wherein the identified protein is selected from: actin, gamma-enteric smooth muscle, kininogen-2 (isoform II), serpin A3-3, serpin A3-5, CD40 ligand, C-type lectin domain family 11 member A, talin-1, Ig heavy chain MemS-like, partial, SERPINA3-8, protein S100-A10 , mitochondrial fission 1 protein, factor XIIa inhibitor, sphingomyelin phosphodiesterase, pleckstrin, keratin, type II cytoskeletal 7, collectin-43, serpin A3-7, ras-related protein Rab-6B, proheparin-binding EGF-like growth factor, tRNA guanosine-2′-O-methyltransferase TRM13 homolog, asporin, biglycan, zinc finger and BTB domain-containing protein 48, nucleolar GTP-binding protein, aspartyl-tRNA synthetase, cytoplasmic solute carrier family 2_facilitated glucose transporter member 4, leucine-rich repeat transmembrane neuronal protein 4, toll-like receptor 6, v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog, coiled-coil domain-containing protein 147, transmembrane and TPR repeatcontaining protein 3, cylicin-1, GPI ethanolamine phosphate transferase 2, AFG3-like protein 2, dynamin-2, nebulette, structural maintenance of chromosomes prot

Assignees

• Univ California

Inventors

• Brooks George A
• Hussien Rajaa
• Rihn Bertrand

Classifications

• C12N2500/84

  from mammals · CPC title

• Y10S977/773

  Nanoparticle, i.e. structure having three dimensions of 100 nm or less · CPC title

• G01N33/6848

  Methods of protein analysis involving mass spectrometry · CPC title

• B82Y5/00

  Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery · CPC title

• G01N2560/00

  Chemical aspects of mass spectrometric analysis of biological material · CPC title