Chemical dissociation of cell aggregates

Abstract

The invention provides a novel method of dissociating anchorage independent and dependent cell aggregates. The invention also includes the cells resulting from the methods of the invention and the use of the cells in various applications requiring the generation of a single cell suspension.

1 . A method for chemically dissociating cell aggregates in a medium comprising the steps of: 
 a) increasing the pH of the medium;    b) generating a single cell suspension; and    c) decreasing the pH of the medium.    
   
   
       2 . The method of  claim 1 , wherein generating the single cell suspension comprises gently pipetting the cell aggregates.  
   
   
       3 . The method of  claim 1 , further comprising (d) gently pipetting the single cell suspension after decreasing the pH.  
   
   
       4 . The method of  claim 1 , wherein the increase in pH is provided by alkaline medium.  
   
   
       5 . The method of  claim 4  wherein the alkaline medium comprises medium and a base.  
   
   
       6 . The method of  claim 5 , wherein the base is selected from the group consisting of sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, zinc hydroxide, sodium phosphate tribasic dodecahydrate and sodium carbonate.  
   
   
       7 . The method of  claim 1 , wherein the pH is decreased by placing the cells in fresh medium which is at neutral pH.  
   
   
       8 . The method of  claim 1 , wherein the decrease in pH is provided by acidic medium.  
   
   
       9 . The method of  claim 8 , wherein the acidic medium comprises medium and an acid.  
   
   
       10 . The method of  claim 9 , wherein the acid is selected from the group consisting of hydrochloric acid, sulphuric acid, acetic acid, citric acid, phosphoric acid, and carbonic acid.  
   
   
       11 . The method of  claim 1 , wherein generating the single cell suspension is for a finite period of time.  
   
   
       12 . The method of  claim 11 , wherein the finite period of time is between about 7 and 10 minutes.  
   
   
       13 . The method of  claim 2 , wherein the gentle pipetting is after about 2 minutes and after about 5 minutes.  
   
   
       14 . The method of  claim 13 , wherein gentle pipetting is repeated 5 to 10 times at each time point.  
   
   
       15 . The method of  claim 2 , wherein gentle pipetting is repeated 5 to 10 times.  
   
   
       16 . The method of  claim 1 , wherein pH is increased to a pH ranging from 9 to 11.5.  
   
   
       17 . The method of  claim 1 , wherein the pH is increased to about 10.  
   
   
       18 . The method of  claim 1 , wherein the pH is decreased to a pH ranging from 7.0 to 8.5.  
   
   
       19 . The method of  claim 1 , wherein the pH is decreased to about 8.1.  
   
   
       20 . The method of  claim 1 , wherein the cell aggregates are selected from the group consisting of two cells, primary tissue, spheres of cells and clusters of cells.  
   
   
       21 . The method of  claim 1 , wherein the cell aggregates are pieces of animal tissue.  
   
   
       22 . The method of  claim 1 , wherein the cell aggregates are produced in in vitro cultures.  
   
   
       23 . The method of  claim 1 , wherein the cell aggregates are mammalian.  
   
   
       24 . The method of  claim 1 , wherein the cell aggregates are selected from the group consisting of neurospheres, hematopoietic cells, mammary epithelial cells, pancreatic cells and cell lines.  
   
   
       25 . The method according to  claim 24 , wherein the cell lines are CHO cells or BHK cells.  
   
   
       26 . A method for chemically detaching cells from a physical surface comprising the steps of: 
 a) increasing the pH of the medium in the presence of an chelating agent;    b) generating a single cell suspension; and    c) decreasing the pH of the medium.    
   
   
       27 . The method of  claim 26 , wherein the chelating agent is EDTA.  
   
   
       28 . The method of  claim 26 , wherein the cells are rocked in a culture vessel after the increase in pH.  
   
   
       29 . The method of  claim 26 , wherein generating a single cell suspension comprises pipetting the medium over the physical surface.  
   
   
       30 . The method of  claim 29 , wherein gentle pipetting is repeated 5 to 10 times.  
   
   
       31 . The method of  claim 26 , further comprising (d) centrifuging the single cell suspension prior to decreasing the pH of the medium.  
   
   
       32 . The method of  claim 26 , wherein the increase in pH is provided by alkaline medium.  
   
   
       33 . The method of  claim 32 , wherein the alkaline medium comprises medium and a base.  
   
   
       34 . The method of  claim 33 , wherein the base is selected from the group consisting of sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, zinc hydroxide, sodium phosphate tribasic dodecahydrate and sodium carbonate.  
   
   
       35 . The method of  claim 32 , wherein the alkaline medium comprises phosphate buffered saline and a base.  
   
   
       36 . The method of  claim 35 , wherein the base is sodium hydroxide.  
   
   
       37 . The method of  claim 26 , wherein the pH is increased to a pH ranging from 9 to 11.5.  
   
   
       38 . The method of  claim 26 , wherein the pH is increased to about 10.  
   
   
       39 . The method of  claim 26 , wherein the pH is decreased by placing the cells in fresh medium which is at neutral pH.  
   
   
       40 . The method of  claim 26 , wherein the decrease in pH is provided by acidic medium.  
   
   
       41 . The method of  claim 40 , wherein the acidic medium comprises medium and an acid.  
   
   
       42 . The method of  claim 41 , wherein the acid is selected from the group consisting of hydrochloric acid, sulphuric acid, acetic acid, citric acid, phosphoric acid, and carbonic acid.  
   
   
       43 . The method of  claim 40 , wherein the acidic medium comprises phosphate buffered saline and an acid.  
   
   
       44 . The method of  claim 43 , wherein the acid is hydrochloric acid.  
   
   
       45 . The method of  claim 26  wherein the pH is decreased to a pH ranging from pH 7.0 to pH 8.5.  
   
   
       46 . The method of  claim 45 , wherein the pH is decreased to about 8.1.  
   
   
       47 . The method of  claim 26 , wherein the step of generating the single cell suspension is for a finite period of time.  
   
   
       48 . The method of  claim 47 , wherein the finite period of time is between 15 to 25 minutes.  
   
   
       49 . The method of  claim 45 , further comprising rocking the cells gently for 2 to 5 minutes to detach the cells.  
   
   
       50 . The method of  claim 45 , further comprising pipetting the cells after 15 to 25 minutes to dissociate cells into single cell suspension.  
   
   
       51 . The method of  claim 26 , wherein the cells are selected from the group consisting of two cells, single cells, clusters of cells, monolayers and multiple layers.  
   
   
       52 . The method of  claim 26 , wherein the cells are in in vitro cell culture.  
   
   
       53 . The method of  claim 26 , wherein the cells are mammalian.  
   
   
       54 . The method of  claim 26 , wherein the cells are selected from the group consisting of Chinese Hamster Ovary cells, L-929 murine lung fibroblast cells, murine mammary epithelial cells MRC-5, He La, Vero, and MDCK.  
   
   
       55 . Chemically dissociated cells resulting from the method of  claim 1 .  
   
   
       56 . The use of the cells according to  claim 55  for in vitro cell culture.  
   
   
       57 . The use of the cells according to  claim 55  for cellular therapy.  
   
   
       58 . The use of the cells according to  claim 55  for production of bio-molecules.  
   
   
       59 . The use of the cells according to  claim 55  for diagnostic analysis.  
   
   
       60 . The use of the cells of  claim 55  for cell analysis.  
   
   
       61 . The use of  claim 60  for molecular, phenotype or functional analysis.  
   
   
       62 . Chemically detached cells resulting from the method of  claim 26 .  
   
   
       63 . The use of the cells according to  claim 62  for in vitro cell culture.  
   
   
       64 . The use of the cells according to  claim 62  for cellular therapy.  
   
   
       65 . The use of the cells according to  claim 62  for production of bio-molecules.  
   
   
       66 . The use of the cells according to  claim 62  for diagnostic analysis.  
   
   
       67 . The use of the cells of  claim 62  for cell analysis.  
   
   
       68 . The use of  claim 67  for molecular, phenotype or functional analysis.  
   
   
       69 . A kit for generating chemically dissociated cells comprising: 
 a) Alkaline medium;    b) Acidic medium; and    d) Instructions for use.    
   
   
       70 . A kit for chemically detaching cells from a physical surface comprising: 
 a) Alkaline medium;    b) A chelating agent;    c) Acidic medium; and    d) Instructions for use.