Differentiation of ipscs in bioreactors
AssigneeRepairon GmbH
InventorLuis HAUPT
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Abstract
The present invention relates to a method of producing a population of differentiated cells from pluripotent stem cells (PSC) in suspension culture in a bioreactor, the method comprising (i) cultivating the PSC under suitable conditions to allow mesodermal induction, (ii) inducing differentiation under suitable conditions of the PSC of step (i), and (iii) conducting metabolic selection under suitable conditions of the PSC of step (ii), wherein steps (i) to (iii) are conducted in a closed bioreactor system, thereby producing a population of differentiated cells. The present invention further relates to a population of differentiated cells obtainable or obtained by said method. The present invention further describes a method of dissociating cell aggregates in a closed bioreactor system.
Claims16
A method of producing a population of differentiated cells from pluripotent stem cells (PSC) in suspension culture in a bioreactor, the method comprising
- (i) cultivating the PSC under suitable conditions to allow mesodermal induction,
- (ii) inducing differentiation under suitable conditions of the PSC of step (i), and
- (iii) optionally selecting the PSC of step (ii),
- wherein steps (i) to (iii) are conducted in a closed bioreactor system, thereby producing a population of differentiated cells.
The method of any one of the preceding claims, wherein after step (iii) a further step (iv) is conducted of recovery of the PSC of step (iii) under suitable conditions in the closed bioreactor system.
The method of any one of the preceding claims, wherein after step (ii), (iii) or (iv) a further step (v) is conducted of harvesting of the population of differentiated cells.
The method of any of the preceding claims, wherein the differentiated cells are cardiomyocytes.
The method of any one of the preceding claims, wherein enriching in step (iii) comprises conducting metabolic selection under suitable conditions.
The method of any one of the preceding claims, wherein the closed bioreactor system is a stirred bioreactor, a rocking motion bioreactor and/or a multi parallel bioreactor.
The method of any one of the preceding claims, wherein
- (a) the medium is
- in step (0) iPS-brew basal medium comprising iPS-brew supplement;
- in step (i) a mesodermal induction medium (MIM), which comprises RPMI-1640, about 1 to 5% B27 minus insulin, about 100 to 300 μmol/L I-ascorbic acid-2-phosphate sesquimagnesium salt hydrate, about 0.1 to 10 mM sodium pyruvate, about 5 to 15 ng/ml Activin A, about 1 to 10 ng/ml BMP4, about 1 to 10 ng/ml bFGF, optionally about 1 to 3 μM CHIR99021;
- in step (ii) a cardiomyocyte induction medium (CM-IM) comprising RPMI-1640, about 1 to 5% B27, about 100 to 300 μmol/L I-ascorbic acid-2-phosphate sesquimagnesium salt hydrate, about 0.1 to 10 mM sodium pyruvate, about 1 to 15 μM IWP4;
- in step (iii) a selection medium comprising RPMI-1640 minus glucose, about 1 to 5 mM Lactate, about 0.01 to 0.5 mM 2-Mercaptoethanol, about 2 to 8 mM HEPES;
- and
- in step (iv) a basal serum-free medium (BSFM), which comprises RPMI-1640, about 1 to 5% B27, about 100 to 300 μmol/L L-ascorbic acid-2-phosphate sesquimagnesium salt hydrate, and about 0.1 to 10 mM sodium pyruvate;
- and/or
- (b) the steps are conducted for the following periods
- step (0) optionally about 4 to 6 days,
- step (i) about 24 hours to 5 days,
- step (ii) about 4 to 10 days,
- step (ii) (a) about 1 to 5 days,
- step (iii) about 5 to 10 days,
- step (iv) about 12 hours to 5 days.
The method of any one of the preceding claims, wherein a change of the medium is conducted at least at the transition from step (0) to step (i), from step (i) to step (ii), from step (ii) to step (ii) (a), from step (ii) (a) to step (iii), and from step (iii) to step (iv), wherein the change of the medium preferably includes a step of washing the cells comprised in the closed bioreactor system.
The method of any one of the preceding claims, wherein the medium change is a complete or a partial medium change;
- wherein the change of medium from step (0) to step (i) comprises a partial medium change, preferably wherein the medium of step (0) remains unchanged for an amount of about 5 to 75% v/v, preferably 25% v/v;
- and/or
- wherein step (0), step (ii), step (ii) (a), step (iii) and step (iv) are conducted comprising perfusion of the medium.
The method of any one of the preceding claims, wherein cell aggregates formed during step (0) are dissociated within the closed bioreactor system.
The method of any one of the preceding claims, wherein the differentiated cells are selected from the group consisting of cardiomyocytes, skeletal muscle cells, fibroblasts, stromal cells, endothelial cells, and leukocytes.
Dependencies are inferred from the claim text.