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AOF medium for MSC culture

RSTEM

Product name: R:STEM Medium for hMSC High Growth(Ref. No. EM1-500)
FIRM

(Applicable areas: Japan)

AOF (animal origin-free) medium for the first time in Japan with FIRM Mark certification (certification No.: CAM-0001) based on ISO 20399*1 as an applicable standard

General requirements for ancillary materials used in the manufacture of cell therapy products and gene therapy products

R:STEM Medium for hMSC High Growth (Ref. No. EM1-500)

Certified products are limited to those manufactured in Japan.

Rohto Pharmaceutical's complete AOF media, “R:STEM” has become the first in Japan to receive FIRM Mark certification. The certification ceremony was held on May 17, 2024. Click here for more details.

AOF medium
  • Reduction in the risk of viruses caused by the use of serum
    Not containing human/animal-derived raw materials
  • Reduction in the risk of changes in cell characteristics caused by EVs
    Medium not containing EVs
  • Reduction in cellular toxicity, including reduced cell proliferative capacity
    Not containing antibiotics
  • Reduction in the time required for lot checks
    Low variability due to the use of chemically definable ingredients
  • Reduction in the time and effort of pretreatments such as addition of supplements
    Ready-to-use medium for immediate use after thawing
Strict quality control in every step of media production
Features
  • Obtained FIRM Mark certification based on ISO 20399 as an applicable standard
  • Obtained qualification certificate for materials of regenerative, cellular, and gene therapy products
  • Serum-free media free of animal- and human-derived ingredients
  • Primary culture capability
  • Excellent culture performance of hMSCs (adipose derived and umbilical cord derived)
  • Small lot-to-lot difference
  • EVs content in the medium is below the detection limit
  • Antibiotics free
  • Ready-to-use medium for immediate use after thawing

Measurement of the amount of EVs contained in the medium

Results

The amount of EVs in R:STEM (EM-500) is not more than the detection limit.

Measurement of the amount of EVs contained in the medium
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The amount of EVs contained in R:STEM (EM1-500) or commercial xeno-free medium was measured using CD9/CD63 ELISA kit.

(Mann-Whitney U test, *p<0.05)

Stability of cell culture using R:STEM

Results

R:STEM (EM1-500) has a small lot-to-lot variability, which minimizes variability in cell culture.

Stability of cell culture using R:STEM(PDL)
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Stability of cell culture using R:STEM(PDT)
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Adipose-derived hMSCs were cultured in three lots of R:STEM (EM1-500) from Passage 2 to Passage 4, and the cell proliferation capacity was evaluated based on the population doubling level (PDL) and population doubling time (PDT) calculated over time for cells in Passage 4.

Investigation of cell proliferation capacity of adipose-derived hMSCs

Results

R:STEM (EM1-500) causes proliferation of adipose-derived hMSCs.

Investigation of cell proliferation capacity of adipose-derived hMSCs(PDL)
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Investigation of cell proliferation capacity of adipose-derived hMSCs(PDT)
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Adipose-derived hMSCs were cultured using R:STEM(EM1-500), commercially available AOF medium, or commercially available xeno-free medium from Passage 2 to Passage 4, and the cell proliferation capacity was evaluated based on the population doubling level (PDL) and population doubling time (PDT) calculated over time for cells, and the cell status was observed.

(Kruskal-Wallis test, n.s.)

Day 10: Passage 4, photo: commercial AOF medium Day 10: Passage 4, photo: commercial xeno-free medium Day 10: Passage 4, photo: R:STEM
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Analysis of hMSC-related surface markers of adipose-derived hMSCs

Results

Adipose-derived hMSCs cultured in R:STEM (EM1-500) meet the criteria for hMSCs specified by the International Society for Cell Therapy.

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Adipose-derived hMSCs were cultured in R:STEM (EM1-500) from Passage 2 to Passage 4, and MSC-related surface markers (positive markers CD73, CD90, and CD 105: >95%; negative markers CD11b, CD34, CD45, and HLADR: <2%) specified by the International Society for Cell Therapy were analyzed by flow cytometry for cells in Passage 4.

Investigation of differentiation potential of adipose-derived hMSCs

Results

Adipose-derived hMSCs cultured in R:STEM (EM1-500) have the trilineage differentiation ability.

Fat differentiation Bone differentiation Cartilage differentiation
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Adipose-derived hMSCs were cultured in R:STEM (EM1-500) from Passage 2 to Passage 4, and differentiation of cells in Passage 4 into fat, bone, and chondrocytes was induced. Differentiation was assessed by oil red O staining, alizarin red staining, and alcian blue staining.

Karyotyping of adipose-derived hMSCs

Results

R:STEM (EM1-500) does not affect the chromosomes of adipose-derived hMSCs.

Chromosome mitotic images Karyogram
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Adipose-derived hMSCs were cultured in R:STEM (EM1-500) from Passage 2 to Passage 4, and after staining of the cells in Passage 4 by G-banding, karyotyping was performed on eight cells selected randomly.

Evaluation of EVs production ability of adipose-derived hMSCs

Results

R:STEM (EM1-500) promotes the EVs production ability of adipose-derived hMSCs.

Evaluation of EVs production ability of adipose-derived hMSCs/EV production per cell (pg)
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Evaluation of EVs production ability of adipose-derived hMSCs/Number of EVs secreted per cell
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Adipose-derived hMSCs were cultured in R:STEM (EM1-500) or commercially available AOF media from Passage 2 to Passage 4, and the amount of EVs contained in the culture supernatant was measured by CD9/CD63 ELISA kit and ExoCounter when cells from Passage 4 were cultured.

(Mann-Whitney U test, *p<0.05)

Evaluation of HGF production ability of adipose-derived hMSCs

Results

R:STEM (EM1-500) promotes the HGF production ability of adipose-derived hMSCs.

Evaluation of HGF production ability of adipose-derived hMSCs
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Adipose-derived hMSCs were cultured in R:STEM (EM1-500), commercially available AOF media, or commercially available xeno-free medium from Passage 2 to Passage 4, and the amount of HGF contained in the culture supernatant was measured by HGF ELISA kit when cells from Passage 4 were cultured .

(Steel's test, *p<0.05)

Investigation of cell proliferation capacity of umbilical cord–derived hMSCs

Results

R:STEM (EM1-500) causes proliferation of umbilical cord–derived hMSCs.

Investigation of cell proliferation capacity of umbilical cord–derived hMSCs
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Umbilical cord–derived hMSCs were cultured using R:STEM (EM1-500), commercially available AOF medium, or commercially available xeno-free medium from Passage 2 to Passage 4, and the cell proliferation capacity was evaluated based on the population doubling level (PDL) calculated over time for cells, and the cell status was observed.

(Kruskal-Wallis test, n.s.)

Day 10: Passage 4, photo: Competitor A (commercial AOF medium) Day 10: Passage 4, photo: Competitor B (commercial xeno-free medium) Day 10: Passage 4, photo: R:STEM
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Analysis of hMSC-related surface markers of umbilical cord–derived hMSCs

Results

Umbilical cord–derived MSCs cultured in R:STEM (EM1-500) meet the criteria for hMSCs specified by the International Society for Cell Therapy.

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Umbilical cord–derived hMSCs were cultured in R:STEM (EM1-500) from Passage 2 to Passage 4, and MSC-related surface markers (positive markers CD73, CD90, and CD 105: >95%; negative markers CD11b, CD34, CD45, and HLADR: <2%) specified by the International Society for Cell Therapy were analyzed by flow cytometry for cells in Passage 4.

Evaluation of EVs production ability of umbilical cord–derived hMSCs

Results

R:STEM (EM1-500) promotes the EVs production ability of umbilical cord–derived hMSCs.

Evaluation of EVs production ability of umbilical cord–derived hMSCs/EV production per cell (pg)
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Evaluation of EVs production ability of umbilical cord–derived hMSCs/Number of EVs secreted per cell
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Umbilical cord–derived hMSCs were cultured in R:STEM (EM1-500) or commercially available AOF media from Passage 2 to Passage 4, and the amount of EVs contained in the culture supernatant was measured by CD9/CD63 ELISA kit and ExoCounter when cells from Passage 4 were cultured.

(Mann-Whitney U test, *p<0.05)

Evaluation of HGF production ability of umbilical cord–derived hMSCs

Results

R:STEM (EM1-500) promotes the HGF production ability of umbilical cord–derived hMSCs.

Evaluation of HGF production ability of umbilical cord–derived hMSCs
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Umbilical cord–derived hMSCs were cultured in R:STEM (EM1-500) or commercially available AOF media, and commercially available Xeno-free medium from Passage 2 to Passage 4, and the amount of HGF contained in the culture supernatant was measured by HGF ELISA kit when cells from Passage 4 were cultured.

(Steel's test, *p<0.05)

※All the data presented was obtained by our company.