Cellular test systems for the determination of the biological activities of neurotoxin polypeptides

Patent metadata

Field	Value
Publication number	US-10125350-B2
Application number	US-201314435583-A
Country	US
Kind code	B2
Filing date	Oct 15, 2013
Priority date	Oct 16, 2012
Publication date	Nov 13, 2018
Grant date	Nov 13, 2018

Abstract

The present invention pertains to a method for the generation of neurotoxin-sensitive, neuronal differentiated cells comprising the steps of: a) cultivating tumor cells which are able to differentiate into neuronal cells in a culture medium under conditions and for a time which primes said tumor cells for neuronal differentiation; and b) cultivating the tumor cells primed for neuronal differentiation of a) in a differentiation medium having an osmolality of 100 to 270 mOsm/kg, and comprising (i) B27 supplement and/or (ii) N2 supplement, for at least 3 days, thereby obtaining neurotoxin-sensitive, neuronal differentiated cells. The invention further relates to neurotoxin-sensitive, neuronal differentiated cells obtainable by the method of the invention. In addition, the invention encompasses a method for determining the activity of a neurotoxin polypeptide comprising the steps of: a) contacting the neurotoxin-sensitive, neuronal differentiated cells obtainable by the method of the invention with a neurotoxin polypeptide; b) cultivating the neurotoxin-sensitive, neuronal differentiated cells of step a) for 3 to 74 hours or 72 hours under conditions which allow for the neurotoxin polypeptide to exert its biological activity; and c) determining the activity of the neurotoxin polypeptide in the said cells after cultivation according to step b). Finally, the invention provides for a medium comprising OptiMEM, FBS, B27 supplement, and N2 supplement.

First claim

The invention claimed is:

1. A method for the generation of neurotoxin-sensitive, neuronal differentiated cells comprising the steps of:
a) cultivating SiMa tumor cells in a culture medium under conditions and for a period of time which primes the SiMa tumor cells for neuronal differentiation, wherein the culture medium comprises 80 to 98.8% OptiMEM, 1 to 10% Fetal Bovine Serum (FBS), 0.2 to 5% B27 supplement and/or 0.2 to 5% N2 supplement and, optionally, non-essential amino acids and/or an antibiotic;
b) adjusting the osmolality of a differentiation medium comprising (i) B27 supplement and/or (ii) N2 supplement to an osmolality of 180 to 230 mOsm/kg, wherein the differentiation medium comprises 78 to 98.3% neurobasal medium, 1 to 10% FBS, 0.5 to 2% GlutaMAX, 0.2 to 5% B27 supplement, and/or 0.2 to 5% N2 supplement; and
c) cultivating the SiMa tumor cells primed for neuronal differentiation in step a) for at least 3 days in the differentiation medium of step b) having an osmolality of 180 to 230 mOsm/kg,
whereby the sensitivity to Clostridium neurotoxin in the neuronal differentiated cells cultivated according to steps a) and c) is increased compared to SiMa tumor cells cultivated in a differentiation medium having an osmolality of about 290-350 mOsm/kg.

2. The method of claim 1 , wherein the differentiation medium further comprises retinoic acid.

3. The method of claim 2 , wherein the retinoic acid is present in a concentration of between 0.01 μM and 300 μM.

4. The method of claim 1 , wherein the differentiation medium further comprises an antibiotic agent and/or a cytostatic agent which inhibits growth of non-neuronal cells.

5. The method of claim 1 , wherein the differentiation medium further comprises ganglioside GT1b (GT1b).

6. The method of claim 5 , wherein the GT1b is present in a concentration of between 25 μM and 75 μM or is present in a concentration of 50 μM.

7. The method of claim 1 , wherein step a) comprises reduction of serum from the culture medium and/or addition of retinoic acid.

8. The method of claim 1 , wherein the tumor cells are SiMa cells (DSMZ ACC deposit number: 164).

9. The method of claim 8 , wherein the SiMa cells are cultivated in step a) for a period of at least 36 hours.

10. The method of claim 8 , wherein the culture medium in step a) further comprises an antibiotic agent and/or non-essential amino acids (NEAA).

11. The method of claim 8 , wherein the SiMa cells are cultivated in step a) on tissue culture dishes which are coated with at least one compound selected from the group consisting of poly-L-lysine, poly-D-lysine, collagen, laminins, and gelatin.

12. The method of claim 1 , wherein the osmolality of the differentiation medium is about 225 mOsm/kg.

Assignees

• Merz Pharma Gmbh & Co Kgaa

Inventors

• Eisele Karl-Heinz
• Harting Kai

Classifications

• C12N5/0619 Primary

  Neurons · CPC title

• C12N2500/38

  Vitamins · CPC title

• C12N2506/30

  from cancer cells, e.g. reversion of tumour cells · CPC title

• C12N2500/60

  Buffer, e.g. pH regulation, osmotic pressure · CPC title

• G01N33/573

  for enzymes or isoenzymes · CPC title