1. Thaw MesenGro Supplement. It is recommended to thaw overnight at 2 to 8°C. Thawed material can be aliquoted and
stored at -80°C, but further freeze-thaw cycles should be avoided.
2. To make 100 mL MesenGro complete medium, aseptically add 10 mL MesenGro Supplement to 90 mL MesenGro Basal
3. Antibiotics such as the Pen-Strep solution can also be added to the complete medium if desired. We recommend the final
concentrations of 100 units/mL Penicillin and 100 microgram/mL Streptomycin.
Once made, MesenGro complete medium (basal, supplement and L-glutamine) is stable for 1 month when stored in the dark
at 2 to 8°C.
|Country of Origin||USA|
The MesenGro® Chemically Defined MSC Medium (for simplicity, referred to as “MesenGro” in this document) has been
designed for the maximum expansion of human mesenchymal stem cells (MSCs) derived from bone marrow, cord blood, or
MesenGro is chemically defined as its components are either chemically synthesized, recombinantly produced and purified. It
can be considered xeno-free as none of its ingredients are directly derived from non-human animals. MesenGro further offers
the convenience of no plate-coating if used with certain tissue culture vessels (see below), saving cost of coating materials
and time needed for coating.
Content and Storage of the Product
MesenGro (Catalog No. MGro-500) contains two components:
MesenGro Basal Medium
1 x 450mL
2 to 8°C. Protect from light
1 x 50mL
-80°C. Protect from light
Additional key reagents required or suggested
- Without plate-coating: Corning CellBIND® tissue culture flasks (e.g., 25 cm2 tissue culture flasks, Corning cat# 3289)
- With plate-coating: Fibronectin (StemRD cat# Fibro-10 or equivalent) and regular TC-treated tissue culture flasks
- TrypLE™ Express (GIBCO #12604), or Trypsin 0.05%/0.53 mM EDTA (e.g. Mediatech #25-051-CI), or equivalent
MSCs cultured in serum-containing or serum-free media can be quickly and easily adapted into MesenGro. In most cases, a
one-step transition into MesenGro complete medium is sufficient. If so desired, step-wise adaptation with a gradual increase
of the amount of MesenGro (e.g. 20%, 40%, etc) can also be performed.
Recovery of Cryopreserved Human MSCs:
Frozen human MSCs, regardless of what medium was used to grow or freeze them before, can be easily and quickly adapted
into MesenGro. A one-step transition into MesenGro medium as outlined below is generally sufficient for cells that have been
grown and frozen in serum-containing or serum-free media.
1. Rapidly thaw frozen vial of cells in a 37°C water bath.
2. Transfer the cells into a 50 mL conical tube.
3. For every 1 mL of cell suspension, add 10 mL of pre-warmed (37°C) MesenGro complete medium in a drop-wise manner
while gently swirling the tube.
4. Plate the contents of the conical tube into a tissue culture flask or multiple wells of a tissue culture plate. Alternatively, cells
can also be centrifuged at 250 x g (~1200 rpm) for 10 minutes, resuspended in MesenGro complete medium and then plated.
5. Incubate at 36 to 38°C in a humidified atmosphere containing 4 to 6% CO2.
6. After 24 hrs, discard old medium and feed cells with new medium.
7. Maintain the culture by changing medium every 2 days until cell passaging
Passaging MSCs in MesenGro:
Coating of cell culture vessels is NOT necessary when MesenGro is used with certain tissue culture vessels, for example, the
Corning CellBIND® tissue culture products or the BD PrimariaTM tissue culture products (the 25 cm2 tissue culture flasks, BD cat# 353808). Additional brands and models of tissue culture vessels are currently under evaluation. For tissue culture
vessels that have not been evaluated or for those found to be incompatible with the no-coating procedure, we recommend
coating with Fibronectin (StemRD cat# Fibro-10) at 0.5 – 1 microgram / cm2 surface area for 1 hour at 37°C, followed by
plating cells in MesenGro.
1. Visually inspect the stock culture (growing in MesenGro or other medium formulations) under the microscope and confirm that the cells are ready to be sub-passaged. To maintain the optimal growth potential of MSCs, it is recommended that the cells be passaged when they reach 70% confluency. If the MSC culture is allowed to reach a confluency of 80% or higher, they may stop proliferating after the passage. Therefore, IT IS CRUCIAL NOT TO GROW CELLS OVER 80% CONFLENCY under any circumstances!
2. Pre-warm TrypLE™ Express (or 0.05% Trypsin/0.53 mM EDTA) and MesenGro complete medium to 37°C before use.
3. Remove spent (old) medium from the flask using a pipet and discard.
4. Optional: Wash tissue culture surface with DPBS, remove and discard.
5. Add the TrypLE™ Express (or 0.05% Trypsin/0.53 mM EDTA) to the flask, tilt flask to cover all the cells. We recommend
detaching the cells at room temperature.
Individual well of a 6-well plate (~10 cm2)
T25 flask (25 cm2)
Volume of TrypLE Express or Trypsin
6. Observe the cells under a microscope. When cells start to detach, gently tap the side of the vessel to help loosen the
remaining cells. The time required for the cells to detach should be 1 to 3 minutes if the cells have been cultured in MesenGro. Cells grown in serum-containing media will require longer incubation time to detach.
Once all the cells have detached, proceed quickly to the following step. Do not leave cells in TrypLE™ Express or Trypsin for
an extended amount of time after the cells have detached, as this will adversely affect the growth of MSCs.
7. Upon cell detachment, add MesenGro at twice the volume of the trypsin solution used (e.g. 2 mL for a T25 flask). Collect
the cell suspension in a sterile 15 mL conical tube.
Optional: If 0.05% Trypsin/EDTA solution is used to detach the cells, it is desirable to use the Soybean Trypsin Inhibitor to
neutralize trypsin. This is especially necessary if the washing step (step 8) cannot be processed quickly (within 10 minutes).
[This is because serum-free media formulations like MesenGro does not contain antitrypsin, a component found in animal
serum. Unless removed quickly by centrifugation, trypsin can damage the cells and affect growth.] If TrypLE
Express is used
to detach the cells, then it is not necessary to neutralize it according to its manufacture.
8. Centrifuge cells at 1200 rpm (250 x g) for 10 minutes.
9. Aspire and discard as much supernatant as possible. Resuspend cells in pre-warmed MesenGro complete medium. Take
an aliquot from the cell suspension for cell counting.
10. Place cells into tissue culture vessels at a density of 5 x103 cells/cm2. Tilt the vessel a few times to ensure even distribution of cell suspension.
11. Incubate at 37°C in a humidified atmosphere of 4 to 6% CO2.
12. Replace culture medium every 2 days with fresh, pre-warmed MesenGro complete medium.
13. Pass cells when cell confluency reaches 80% (typically at 3 day intervals).
Cryopreservation of Cells in MesenGro
1. Prepare cryopreservation solution by supplementing MesenGro Basal Medium with 10% MesenGro Supplement and 10%
Dimethyl Sulfoxide (DMSO).
2. Pellet cells by centrifugation, resuspend cells in cryopreservation solution to 1.0x106
cells/mL, and transfer to cryovials.
3. Place cryovials in a freezing container (e.g. Nalgene 5100-0001) and place in a -80°C freezer overnight.
4. Transfer cryovials to liquid nitrogen for long-term storage.