Methods for the preparation of fibroblasts

Abstract

The invention relates to a process for generating fibroblasts, more particularly, to the culturing of fibroblasts in large numbers and of the heterogenic type. The invention is also directed to the use of fibroblasts in the preparation of heterotypic spheroids and a process for the preparation of such heterotypic spheroids.

What is claimed: 
     
         1 . A process for the preparation of fibroblasts comprising the steps of:
 a) Providing a cell-containing tissue sample;   b) Preparing a suspension of primary cells from the cell-containing tissue sample;   c) Culturing the suspension of primary cells wherein fibroblast cell nests are generated from and within the suspension of primary cells;   d) Separating the fibroblast cell nests of step (c) from the suspension of primary cells;   e) Repeating steps (c) to (d) at least once.   
     
     
         2 . A process according to  claim 1 , wherein the suspension of primary cells of step b) is also treated with an enzymatic composition containing one or more enzymes selected from the group consisting of proteases such as serin proteases such as trypsin or dispases, neutral proteases; metalloendopeptidases such as collagenases such as interstitial collagenases and neutrophil collagenases or thermolysin; DNases; hyaloronidases; before culturing according to step c). 
     
     
         3 . A process according to  claim 2 , wherein the enzymatic composition also contains a serum-free medium selected from the group consisting of RPMI, DMEM, F15, MEM, BMEEARL, HAMFSF-12, Leibovitz L-15, McCoys 5A, medium 199, Waymouth medium and HANK-solution. 
     
     
         4 . A process according to  claim 1 , wherein the cell containing tissue sample is either benign tissue such as gastric tissue, colorectal tissue, liver tissue, lung tissue, mucosal tissue, cerebral tissue, pancreas tissue, hepatic tissue, dermal tissue, prostate or periprostatic tissue, gastric tissue, colonic tissue, ovarial tissue, breast tissue, cervical tissue or glioma tissue or malign tissue such as inflammatory tissue, metastatic or tumour tissue such as tumour tissue from gastric, pancreas, colorectal, liver, lung, breast, cervical, mucosal, cerebral, hepatic dermal, colonic, ovarial, sarcoma, prostate or glioma tumours. 
     
     
         5 . A process according to  claim 1 , wherein the growth medium comprises a buffer, a serum, an antibiotic and/or a fungicide. 
     
     
         6 . A process according to  claim 5 , wherein the buffer is phosphate buffered saline (PBS), the serum is foetal calf or bovine serum (FCS or FBS), the antibiotic is Cefazoline 2.0 and the fungicide is amphotericin B. 
     
     
         7 . A process according to  claim 1 , wherein the tissue is incubated in the growth medium at 37° C. for at least 3 days. 
     
     
         8 . A process according to  claim 7 , wherein the suspension of primary cells is cultured in a vessel coated with a gelatine solution. 
     
     
         9 . A process according to  claim 1 , wherein the culture medium comprises DMEM and FCS or FBS. 
     
     
         10 . A process according to  claim 1 , wherein step c) is repeated at least 3 times. 
     
     
         11 . A process according to  claim 1 , wherein step c) is repeated at least 5 times. 
     
     
         12 . Fibroblasts, obtained by the process as defined by  claim 1 . 
     
     
         13 . A process for the preparation of multi-cellular spheroids comprising:
 a) Preparing a suspension of single cells from a biological tissue or cell-containing bodily fluid;   b) Adding fibroblast cells as defined in  claim 12  to the suspension;   c) Adjusting the concentration of the cells in the suspension to a concentration in the range of from 10 3  cells to 10 7  cells;   d) Adding 2 to 50 vol.-% of an inert matrix to the suspension of single cells; and   e) Incubating the suspension of single cells under conditions suitable for the formation of spheroids.   
     
     
         14 . A process according to  claim 13 , wherein the tissue is either a healthy tissue, a tumour tissue, a benign or malignant primary tissue. 
     
     
         15 . A process according to  claim 13 , further comprising the step: treating the suspension of single cells to remove dead and/or dying cells and/or cell debris. 
     
     
         16 . A process according to  claim 13 , wherein the tissue is a mammalian tissue. 
     
     
         17 . A process according to  claim 13 , wherein the tissue is treated mechanically before preparing the suspension of single cells. 
     
     
         18 . A process according to  claim 13 , wherein the single cell suspension is prepared in a medium comprising serum, buffer, interleukins, chemokines, growth factors, hydrogen carbonate, glucose, physiological salts, amino acids and hormones. 
     
     
         19 . A process according to  claim 18 , wherein the medium further comprises at least one antibiotic selected from the group consisting of penicillin, streptomycin, neomycin, ampicillin, metronidazole, ciprofloxacin, gentamicin, Amphotericin B, Kanamycin and Nystatin. 
     
     
         20 . A process according to  claim 13 , wherein the concentration of single cells is adjusted from 10 3  to 10 7  cells/ml medium. 
     
     
         21 . A process according to  claim 13 , wherein the inert matrix is added in an amount of from 2 vol.-% to 50 vol.-% based on the total volume of the medium. 
     
     
         22 . A process according to  claim 13 , wherein the inert matrix is derived from a nonhuman source. 
     
     
         23 . A process according to  claim 22 , wherein the inert matrix is selected from the group comprising cellulose ether, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hypomellose, methyl cellulose, methylethyl cellulose, polyethylene glycol, and agarose. 
     
     
         24 . A process according to  claim 13 , wherein the incubation is performed at about 37° C. in an atmosphere containing from 4 vol.-% to 6 vol.-% CO 2 . 
     
     
         25 . A process according to  claim 24 , wherein the incubation is performed from  4  hours up to 9 days. 
     
     
         26 . A multi-cellular spheroid, obtained by the process as defined by  claim 13 . 
     
     
         27 . Use of the fibroblasts obtained by the process as defined in  claim 1  for the preparation of a multi-cellular spheroid.