Standardized neuronal cell assays from primate species

Abstract

This patent application relates to a method to generate standardized and robust neuronal cell culture assays from primate species, for example suitable for high-throughput screening of drug candidates. The method includes differentiating human and/or non-human primate neuronal precursor cells (NPCs) to neuronal cells (NCs) and producing uniform NC cultures based on a dissociation and reseeding step performed on the differentiated NCs resulting in robust cultures suitable for high-throughput drug screening assays, in particular to screen antisense oligonucleotides.

1 . An in vitro method for producing standardized cell cultures of uniformly distributed differentiated neural cells (NCs) from different primate species, the method comprising the steps of
 a) providing neuronal precursor cells (NPCs);   b) differentiating the NPCs to neural cells (NCs) comprising the steps of
 (i) dissociating the differentiated NCs from its support after about 20 days to about 45 days of differentiation; and 
 (ii) reseeding the cells in a suitable cell culture format and continuing differentiation of the NCs for about 4 days to about 15 days. 
   
     
     
         2 . The method according to  claim 1 , wherein the primate species are selected from the group consisting of human ( Homo sapiens ), Cynomolgus monkey ( Macaca fascicularis ) and Rhesus monkey ( Macaca mulatta ). 
     
     
         3 . The method according to  claim 1 , wherein standardized cell cultures of differentiated NCs are individually produced for 2 primate species, wherein the first primate species is human ( Homo sapiens ) and the second primate species is Cynomolgus monkey ( Macaca fascicularis ). 
     
     
         4 . The method according to any one of  claims 1  to  3 , wherein the NPCs are derived from induced pluripotent stem cells (IPSCs). 
     
     
         5 . The method according to  claim 1 , wherein step b)(i) comprises cultivating the cells in basal medium comprising Shh, FGF8 and ascorbic acid phosphate for a period of about 5 to about 10 days followed by cultivating the cells in basal medium comprising BDNF, GDNF, cAMP and ascorbic acid phosphate for a further period of about 15 days to about 35 days prior to dissociating the differentiated NCs from its support. 
     
     
         6 . The method according to  claim 1 , wherein step b)(i) comprises dissociating the cells with a cell detachment solution. 
     
     
         7 . The method according to  claim 6 , wherein the cell detachment solution is Accutase. 
     
     
         8 . The method according to  claim 1 , wherein the cells are reseeded in 96 well or 384 well plates. 
     
     
         9 . The method according to any one of  claims 1 , wherein step b)(ii) comprises continuing differentiation by cultivating the cells in basal medium supplemented with BDNF, GDNF, cAMP and ascorbic acid after reseeding the cells. 
     
     
         10 . The method according to any one of  claim 1 , wherein the differentiated NCs are essentially uniformly distributed over the cell culture area as assessed by cell nucleus staining. 
     
     
         11 . Use of the cell cultures obtained according to  claim 1  for in vitro testing of the toxicity of a drug candidate. 
     
     
         12 . Use of the cell cultures obtained according to  claim 1  for in vitro testing of the efficacy of a drug candidate. 
     
     
         13 . Use of the cell cultures obtained according to  claim 1  for selecting a drug candidate, in particular for selecting a drug candidate for further development. 
     
     
         14 . The cell cultures for use according to any one of  claims 11  to  13  wherein the drug candidate comprises a polynucleotide or targets a specific sequence of a polynucleotide. 
     
     
         15 . The cell cultures for use according to any one of  claims 11  to  13  wherein the drug candidate comprises at least one nucleic acid molecule such as a RNAi agent or an antisense oligonucleotide. 
     
     
         16 . (canceled)