Corning® Synthemax® II and Enhanced Attachment Microcarriers ...

Cell Seeding on Corning Microcarriers
This protocol is intended for mesenchymal stem cell expansion on microcarriers. Optimal
seeding densities will vary depending on cell type, culture media, and specific application.
Therefore, it is recommended to optimize the seeding density for your culture system.
1. Add 4 mL of Corning microcarrier stock suspension (400 mg) to a 15 mL conical tube,
and allow microcarriers to settle (~2 minutes).
2. Using a 5 mL pipet, gently remove the liquid without disrupting the microcarrier pellet.
3. Reconstitute the microcarriers in 10 mL of culture medium, and store the microcarrier
suspension at 37°C until cell seeding.
4. Harvest cells according to your standard protocols, and prepare a suspension of 500,000
to 750,000 cells in 5 mL of culture medium (Note: A cell number of 500,000-750,000 can
be used as a starting point; however, we recommend optimizing the seeding density for
each culture system).
5. Combine the 10 mL microcarrier suspension from step 3 and the 5 mL cell suspension
from step 4 in a disposable 125 mL disposable spinner flask. This will result in a seeding
density of ~3500 to 5000 cells/cm 2 microcarriers.
6. Place spinner flask containing the microcarrier and cell suspension in 37°C/5% CO 2
humidified incubator and gently rock flask to evenly distribute cells and microcarriers.
7. Incubate for 18 to 24 hours under static conditions to allow for cell adhesion (Note:
The duration of the static phase can vary depending on the cell type and culture media.
Therefore, we recommend optimizing it for each culture system).
8. After 18 to 24 hours, bring culture to a final volume of 45 mL with fresh culture medium
and start intermittent agitation (e.g., 30 rpm for 15 minutes every 2 hours).
9. Continue intermittent agitation for 4 to 7 days or until cells reach 70 to 80% confluence
(Note: In our experience, 70 to 80% confluence corresponds to 30,000 to 50,000 cells/cm 2 ).
10. Replenish 50% of the culture medium every 2 to 3 days. When exchanging culture medium,
allow cell-microcarrier suspension to settle 5 minutes, and using a 25 mL pipet, slowly
remove 50% of culture medium, and replace with fresh medium (i.e., 22.5 mL).
Cell Harvest Procedure
1. Using a 25 mL pipet, mix the cell-microcarrier suspension several times to ensure an
even distribution. Remove a 5 to 10 mL cell-microcarrier sample; the volume should
reflect the confluence of your culture to ensure an adequate harvest for meaningful cell
count.
2. Transfer the sample from step 1 to a 15 mL conical tube, and allow microcarriers to
settle (~5 minutes).
3. Gently aspirate spent culture medium being careful not to disturb the cell-microcarrier
pellet.
4. Wash microcarriers 2X with 10 mL of DPBS; gently aspirate DPBS wash as in step 3.
5. Add 5 mL pre-warmed trypsin-EDTA, swirl tube to evenly distribute enzyme and cellmicrocarriers,
and transfer tube to a 37°C incubator for 5 minutes.
6. During trypsinization, prepare a 50 mL conical tube with 1 mL fetal bovine serum (FBS),
and place a 70 µm cell strainer on the top of the tube.
7. After trypsinization, gently mix the culture with a 5 mL pipet to ensure complete cell
detachment from microcarriers ( Note: To confirm cell detachment, transfer an aliquot
of the suspension to a multiple well dish, and observe under a microscope).
8. Filter the cell-microcarrier suspension through the 70 µm filter into FBS to inhibit
further trypsination.
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