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Human Mesenchymal Stem Cells

Isolation - deriving MSCs

What are the sources of Mesenchymal Stem cells (MSCs)?

Mesenchymal Stem cells are fibroblast-like core cells. They can be found in Bone Marrow tissue, or in connective tissues such as Adipose tissue (fat), cartilage, synovial tissue and cord blood tissue.

Do you have a protocol for Mesenchymal Stem cells production?

There are known protocols for producing Mesenchymal Stem cells from the different tissues. The protocols are well-published and the cells can usually be harvested in aesthetic clinics (i.e. liposuctions) and orthopedic wards,` the stages included lysis of cells (mainly fibroblast), connective tissue digestion, pull down of MSC and seeding.

Are there commercially available MSCs?

Most cell companies (i.e. ATCC, PromoCell) have commercial MSCs.

Can I use the NutriStem® MSC XF medium for the isolation step?

NutriStem® MSC XF Basal Medium is suitable to use from the earliest stage. The medium should be used after the lysis step before the seeding to wash the cells. The medium should be equilibrate at the incubator. The complete medium is used in the later stages of the isolation and in the proliferation and seeding of the MSCs.

Does NutriStem® MSC XF medium need any other supplements?

NutriStem® MSC XF medium is composed of two parts - basal medium and a supplement, that together make the complete medium that does not require any additional supplements. A necessary preparation is coating the culture-ware in an attachment solution such as Matrigel®, Fibronectin etc. The medium supports all attachment matrices. Biological Industries has a specific MSC attachment solution.

Is it important to use a serum-free medium in the isolation stage?

MSCs can be cultured in both serum-free and serum-containing media, but it is better to start the serum free system from the earliest stages.

Is the age of the donor tissue relevant?

The older the donor tissue, the older the MSCs produced and thus the shorter life expectancy of the cells.

Is antibiotics necessary for the isolation stage?

In cells isolated from tissues, a clean and sterile environment is very important, thereby antibiotics are needed, in a concentration higher than usual (2 to 10 fold, depending on the tissue source and condition).

Expansion of hMSC

For how long can cells be kept in NutriStem® MSC XF medium without differentiation?

The period of time cells can be cultured depends on the state of the derived cells. The better the quality of isolation, the longer the culture will keep. Commercial lines, for example, will keep between 3-5 passages.

Can cultureware be precoated?

Pre-coating is advisable - cultureware may be precoated and kept in 2-8°C for a week.

How often should the medium be replaced?

In optimal culturing conditions the medium should be completely replaced every 2-4 days.

When should the cells be subcultured?

In optimal culturing conditions the cells should reach 80% confluency and then be subcultured and the seeding density after subculturing should be 5-6 x 103 cells/cm2

Should the precoated cultureware and medium be equilibrated beforehand?

The cultureware and medium should be placed in a 37°C incubator for 30 minutes prior to seeding.

Is there a recommended protocol for preparing coated cultureware?

The coating protocol is an integral part of the culturing protocol. The specific protocol can be found here

Are antibiotics necessary in the culturing stage?

If the post-isolation cells are pathogen free and the working environment is in sterile and clean condition no antibiotics are necessary. In case of pathogen infection, it is possible to add antibiotics to the culture according to the standard protocols of the work place.

Can MSC NutriStem® XF medium substitute an existing serum-free medium?

MSC NutriStem® XF can substitute any other SF medium with no need for adaptation protocols.

Can MSC NutriStem® XF medium substitute an existing serum containing medium?

MSC NutriStem® XF can substitute any other serum-containing medium with no need for adaptation protocols. Since MSCs are multipotent and so not grow very fast, there is no adaptation needed.

How is the multipotency of MSCs determined?

MSCs have typical fibroblast-like morphology. This morphology is kept during their pre-differentiation stage. The cells continue to multiply and keep their morphology. Morphological changes can be observed according to the age of the culture or to stress conditions dependent on the culture environment. The changes should disappear after a medium change. Undifferentiated MSCs express the following markers: CD73, CD90, CD105 and should be CD34 and CD45 negative.

Can undifferentiated MSCs be cryopreserved?

MSCs can be cryopreserved, preferably with a freezing medium optimized for the freezing of MSCs - NutriFreez® D10 Cryopreservation Medium (05-713-1), a serum-free solution with 10% DMSO content. The following protocol should be used

How can MSCs be subcultured?

Subculturing should be performed at 70-80% confluence. The cells should be detached with a designated solution for that purpose. Biological Industries has products based on recombinant Trypsin: Recombinant Trypsin Solution (03-078-1) and Recombinant Trypsin-EDTA Solution (03-079-1). The Trypsin will enable the quick and effective dissociation of the MSCs with a short exposure time - 2-5 minutes