Culturing Stem Cells in 3-D

Requires Potent Media + Supplements

Neuromics is responding to the many challenges our clients face in building 3-D, in-vivo like, cell- based assays. We do this by offering the most potent Media plus Supplements like FGFS.

Here's a protocol for single cell 3-D assays using hMSCs and Hydrogels. It features use of our ISOKine^TM FGF

Images: Cell-centering in cytocompatible microgels enables long-term single-cell 3D culture by preventing cell escape. a) Qualification of Dex-TA microgel crosslinking as a function of the microemulsion flow rate (Qemulsion) and concentration of the H2O2 feed ([H2O2]feed). Blue, green, and red indicate incomplete crosslinking, complete crosslinking, and H2O2 excess, respectively. b,c) Amplex Red assay to quantify the concentration of residual H2O2 ([H2O2]emulsion) in Dex-TA microgel precursor droplets and crosslinked microgels after their retrieval from the diffusion-based crosslinking platform. d) The microencapsulation procedure had no detrimental effect on short-term cell survival. e) Delayed crosslinking resulted in 4 ± 1% cell escape after 7 d of in vitro culture, as compared to 27 ± 5% cell escape when using coupled emulsification and gelation. f) The number of encapsulated cells per microgel tightly followed the Poisson distribution and remained similar throughout long-term (28 d) of in vitro culture, which confirmed that cell centering prevents cell escape. g–i) MSCs encapsulated in delayed enzymatically crosslinked microgels remained viable and metabolically active throughout 28 d of in vitro culture. j) Positive Oil Red O and k) Alizarin Red staining confirmed that l) more than 60% of the microencapsulated MSCs could differentiate into the adipogenic and osteogenic lineage, respectively. Black scale bars: 50 µm, white scale bars: 5 µm. DOI: 10.1002/smll.20160371.

Protocol for Cell Isolation and Expansion: Human MSCs were isolated from fresh bone marrow samples and cultured as previously described. The use of patient material was approved by the local ethical committee of the Medisch Spectrum Twente and informed written consent was obtained for all samples. In short, nucleated cells in the bone marrow aspirates were counted, seeded in tissue culture flasks at a density of 500 000 cells cm−2, and cultured in MSC proliferation medium, consisting of 10% FBS, 100 U mL−1 penicillin, 100 mg mL−1 streptomycin, 1% GlutaMAX, 0.2 × 10−3 m ascorbic acid, and 1 ng mL−1 bFGF (added fresh) in αMEM. Mouse insulinoma MIN6-B1 cells (provided by Dr. P. Halban, University Medical Center, Geneva, Switzerland) were cultured in MIN6 proliferation medium, consisting of 10% (v/v) FBS, 100 U mL−1 penicillin, and 100 mg mL−1 streptomycin, and 71 × 10−6 m 2-mercaptoethanol (added fresh) in DMEM. When cells reached near confluence, the cells were detached using 0.25% Trypsin-EDTA at 37 °C and subsequently subcultured or used for experimentation.

I am at your beck and call to answer questions on our Cell Based Assay Solution. Pete Shuster-CEO and Owner, direct phone: (612) 801-1007 or pshuster@neuromics.com.