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

Corning ® Synthemax ® II and
Enhanced Attachment Microcarriers
for hMSC Expansion
Protocol
Note:
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.
Materials
w Corning Synthemax II polystyrene microcarriers (Corning Cat. No. 3781)
w Corning Enhanced Attachment polystyrene microcarriers (Corning Cat. No. 3779)
w Corning stemgro ® hMSC medium (Corning cellgro ® Cat. No. 40-410-KIT)
w Corning stemgro hMSC supplement (Corning cellgro Cat. No. 40-410-KIT)
w L-glutamine, liquid (Corning cellgro Cat. No. 25-005-CI)
w 5 mL Stripette ® serological pipets (Corning Cat. No. 4487)
w 10 mL Stripette serological pipets (Corning Cat. No. 4488)
w 25 mL Stripette serological pipets (Corning Cat. No. 4489)
w Cell culture grade water, sterile (Corning cellgro Cat. No. 25-055-CM)
w 125 mL disposable spinner flask (Corning Cat. No. 3152)
w 15 mL conical tube (Corning Cat. No. 430052)
w 50 mL conical tube (Corning Cat. No. 430290)
w 70 µm cell strainer (BD Biosciences Cat. No. 352350)
w Dulbecco’s Phosphate Buffered Saline (DPBS) (Corning cellgro Cat. No. 21-031-CV)
w Trypsin-EDTA (Corning cellgro Cat. No. 25-050-CI)
w Fetal Bovine Serum (FBS) (Corning cellgro Cat. No. 35-015-CV)
w 150 mL easy grip polystyrene storage bottle with 45 mm caps (Corning Cat. No. 431175)
Note: All of the following steps should be done under aseptic conditions using proper aseptic
techniques.
Reconstitution of Corning Microcarriers
1. Prepare a microcarrier suspension by transferring dry microcarriers from the shipping
vial to a sterile 150 mL bottle. Using a 10 mL pipet, add 10 mL of sterile water to wash
additional microcarriers out of the vial. Transfer the wash to the 150 mL bottle containing
dry microcarriers. Add sterile water to achieve a final volume of 100 mL.
a. If using the full contents of the microcarrier vial (10g), you may reconstitute the
microcarriers directly in culture medium.
b. If you do not want to reconstitute the full contents of the microcarrier vial, weigh-out
a specified amount and re-sanitize the microcarriers with 70% ethanol for 30 minutes.
Wash 2X with DPBS and 1X with culture medium before cell seeding to ensure
complete removal of ethanol.
2. Invert and swirl bottle several times to ensure complete wetting of all microcarriers.
3. For a 10g vial, this will result in 100 mg/mL microcarrier suspension with the corresponding
surface area of 36 cm 2 /mL.
4. Store Corning ® microcarrier stock suspension at 4°C.

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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9. Wash filter 2X with 2 to 3 mL DPBS ( Note: To confirm no cells remain on the filter,
transfer the filter to 1 well of a 6 well plate, and observe under a microscope).
10. Pipet cell mixture 3 to 5 times to assure single-cell suspension.
11. Count cells, and calculate the total cell number (Note: If cells are used for further culture
or assay, we recommend to use a defined trypsin inhibitor rather than FBS in step 6).
Bead-to-Bead Expansion of Cells
Note: passage-free cell expansion (i.e., no enzymatic harvest) may not be applicable to all cell
types and may require optimization with enzyme for your cell line and media conditions.
1. Add 4 mL of fresh Corning ® microcarrier stock suspension (400 mg) to a 15 mL conical
tube. Allow microcarriers to settle (~2 minutes).
2. Using a 5 mL pipet, gently remove the liquid without disrupting the microcarrier pellet,
and reconstitute the microcarriers in 10 mL of pre-warmed culture medium.
3. Transfer the microcarrier suspension to a new spinner flask, and store at 37°C until cell
seeding.
4. Based upon the cell count, transfer an appropriate volume of the cell-microcarrier suspension
to the pre-warmed microcarriers prepared in step 2. Adjust the final culture volume
to 15 mL (For example, if your cell count is 200,000 cells per mL and you want a seeding
density of 500,000 cells per spinner flask, then transfer 2.5 mL of cell-microcarrier suspension
to fresh microcarriers prepared in step 2. Add an additional 2.5 mL of medium
to bring the final culture volume to 15 mL).
5. Place spinner flask containing microcarriers and cells in 37°C/5%CO 2 humidified
incubator, and gently rock flask to evenly distribute cells and microcarriers.
6. After 18 to 24 hours, bring culture to a final volume of 45 mL and start intermittent
agitation (e.g., 30 rpm for 15 minutes every 2 hours).
7. 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 ).
8. 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).
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