Cationic Surface Charge Combined with Either Vitronectin or Laminin Dictates the Evolution of Human Embryonic Stem Cells/Microcarrier Aggregates and Cell Growth in Agitated Cultures

Lam, Alan Tin-Lun; Li, Jian; Chen, Allen Kuan-Liang; Reuveny, Shaul; Oh, Steve Kah‐Weng; Birch, William R.

doi:10.1089/scd.2013.0645

Cited by 34 publications

(55 citation statements)

References 76 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2b). As in our previous report [36], the aggregates changed in size over time was due to cell proliferation, and subsequently free MC were being engulfed into the pre-existing proliferating hESC on [57,58], IMR90 line showed very low CM yields (0.32 ± 0.2 × 10 6 cells/mL) in a stirred spinner flask under continuous stirring. This low cell yield is attributed to shear sensitivity of cells during differentiation which requires application of intermittent agitation which is problematic in scaling up of processes.…”

Section: Testing Of Cell Expansion and Cardiomyocyte Differentiation supporting

confidence: 69%

Selection of human induced pluripotent stem cells lines optimization of cardiomyocytes differentiation in an integrated suspension microcarrier bioreactor

et al. 2020

Self Cite

View full text Add to dashboard Cite

Background: The production of large quantities of cardiomyocyte is essential for the needs of cellular therapies. This study describes the selection of a human-induced pluripotent cell (hiPSC) line suitable for production of cardiomyocytes in a fully integrated bioprocess of stem cell expansion and differentiation in microcarrier stirred tank reactor. Methods: Five hiPSC lines were evaluated first for their cardiac differentiation efficiency in monolayer cultures followed by their expansion and differentiation compatibility in microcarrier (MC) cultures under continuous stirring conditions. Results: Three cell lines were highly cardiogenic but only one (FR202) of them was successfully expanded on continuous stirring MC cultures. FR202 was thus selected for cardiac differentiation in a 22-day integrated bioprocess under continuous stirring in a stirred tank bioreactor. In summary, we integrated a MC-based hiPSC expansion (phase 1), CHIR99021-induced cardiomyocyte differentiation step (phase 2), purification using the lactate-based treatment (phase 3) and cell recovery step (phase 4) into one process in one bioreactor, under restricted oxygen control (< 30% DO) and continuous stirring with periodic batch-type media exchanges. High density of undifferentiated hiPSC (2 ± 0.4 × 10 6 cells/mL) was achieved in the expansion phase. By controlling the stirring speed and DO levels in the bioreactor cultures, 7.36 ± 1.2 × 10 6 cells/mL cardiomyocytes with > 80% Troponin T were generated in the CHIR99021-induced differentiation phase. By adding lactate in glucose-free purification media, the purity of cardiomyocytes was enhanced (> 90% Troponin T), with minor cell loss as indicated by the increase in sub-G1 phase and the decrease of aggregate sizes. Lastly, we found that the recovery period is important for generating purer and functional cardiomyocytes (> 96% Troponin T). Three independent runs in a 300-ml working volume confirmed the robustness of this process.

show abstract

Section: Testing Of Cell Expansion and Cardiomyocyte Differentiation supporting

confidence: 69%

Selection of human induced pluripotent stem cells lines optimization of cardiomyocytes differentiation in an integrated suspension microcarrier bioreactor

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…This was incubated at 4°C overnight and then rinsed with PBS before further coating. Similarly, the coating of murine LN (Life Technologies, Carlsbad, CA, USA) was achieved by adding 20 μl of 1 mg/ml LN to 20 mg PLL-coated MC in 600 μl PBS, to make a final LN concentration of 33 μg/ml [ 35 ]. The MCs were similarly incubated at 4°C overnight, followed by a rinse with PBS before being used for cell culture.…”

Section: Methodsmentioning

confidence: 99%

“…hPSCs retain pluripotent markers, retain the ability to differentiate into the three primary germ layers, and exhibit normal diploid karyotypes. Recently, a defined polystyrene-based (≈100 μm diameter) MC coated with cationic poly- l -lysine (PLL) polyelectrolyte and laminin (LN) was developed by our group [ 35 ]. This defined matrix supports the expansion of HES3, H7, and IMR90 under continuous agitation from a single-cell seeding solution achieving high cell yields (14-fold, eightfold, and sevenfold cell expansion, respectively) with excellent viability (>90%).…”

Section: Introductionmentioning

confidence: 99%

Conjoint propagation and differentiation of human embryonic stem cells to cardiomyocytes in a defined microcarrier spinner culture

Lam

Chen

et al. 2014

Stem Cell Res Ther

Self Cite

View full text Add to dashboard Cite

IntroductionMyocardial infarction is accompanied by a significant loss of cardiomyocytes (CMs). Functional CMs, differentiated from human embryonic stem cells (hESCs), offer a potentially unlimited cell source for cardiac disease therapies and regenerative cardiovascular medicine. However, conventional production methods on monolayer culture surfaces cannot adequately supply the large numbers of cells required for such treatments. To this end, an integrated microcarrier (MC) bioprocessing system for hESC propagation and subsequent CM differentiation was developed.MethodsProduction of hESC-derived CMs was initially established in monolayer cultures. This control condition was compared against hESC expansion on laminin-coated MC with cationic surface charge, in a stirred serum-free defined culture. Following expansion, the hESC/MC aggregates were placed in a CM differentiation medium, using Wnt signalling modulators in four different culture conditions. This process eliminated the need for manual colony cutting. The final optimized protocol was tested in stirred spinner flasks, combining expansion and differentiation on the same MC, with only media changes during the culture process.ResultsIn the propagation phase, a 15-fold expansion of viable pluripotent HES-3 was achieved, with homogeneous sized aggregates of 316 ± 11 μm. Of the four differentiation conditions, stirred spinner flask cultures (MC-Sp) provided the best controlled aggregate sizes and yielded 1.9 × 106 CM/ml, as compared to 0.5 × 106 CM/ml using the monolayer cultures method: a four-fold increase in CM/ml. Similar results (1.3 × 106 CM/ml) were obtained with an alternative hESC H7 line. The hESC/MC-derived CM expressed cardiac-specific transcription factors, structural, ion channel genes, and exhibited cross-striations of sarcomeric proteins, thus confirming their cardiac ontogeny. Moreover, E-4031 (0.3 μM) prolonged the QT-interval duration by 40% and verapamil (3 μM) reduced it by 45%, illustrating the suitability of these CM for pharmacological assays.ConclusionsWe have demonstrated a robust and scalable microcarrier system for generating hESC-derived CM. This platform is enabled by defined microcarrier matrices and it integrates cell propagation and differentiation within a continuous process, in serum-free culture media. It can generate significant numbers of CM, which are potentially suitable for future clinical therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/scrt498) contains supplementary material, which is available to authorized users.

show abstract

“…No. 910e0003) was added to the microcarriers to release the nuclei of the attached cells and quantified with the use of the Nucleocounter [48].…”

Section: Analysis Of Cell Attachment To and Spreading On Microcarriersmentioning

confidence: 99%

Serum-free media formulations are cell line–specific and require optimization for microcarrier culture

et al. 2015

Self Cite

View full text Add to dashboard Cite

Cationic Surface Charge Combined with Either Vitronectin or Laminin Dictates the Evolution of Human Embryonic Stem Cells/Microcarrier Aggregates and Cell Growth in Agitated Cultures

Cited by 34 publications

References 76 publications

Selection of human induced pluripotent stem cells lines optimization of cardiomyocytes differentiation in an integrated suspension microcarrier bioreactor

Selection of human induced pluripotent stem cells lines optimization of cardiomyocytes differentiation in an integrated suspension microcarrier bioreactor

Conjoint propagation and differentiation of human embryonic stem cells to cardiomyocytes in a defined microcarrier spinner culture

Serum-free media formulations are cell line–specific and require optimization for microcarrier culture

Contact Info

Product

Resources

About