®
USD2978 (2)

1. Introduction ..................................................................................................................................................3
2. Materials and Methods................................................................................................................................3
2.1 Culturing MSCs ......................................................................................................................................3
2.2 Growth in Spinner Cultures ......................................................................................................................3
2.3 Expansion into Bioreactors ......................................................................................................................4
3. Results .........................................................................................................................................................5
3.1 Growth of Cells in Bioreactor ..................................................................................................................5
4. Discussion ....................................................................................................................................................9
5. References ...................................................................................................................................................9
2

Mesenchymal stem cells (MSCs) are self-renewing cells that differentiate into several terminally differentiated cell types. These cells can be isolated from multiple sources, such as bone marrow, adipose tissue, peripheral blood, and other adult tissues 1-6 , making their use in therapeutics promising.
Humanbone-marrowderivedMSCstrypsinizedasasingle-cellsuspensionafter7dayexpansiononSoloHillCollagen microcarriersin3LBioreactor
However, for many stem cell applications, large numbers of cells are needed. Difficulties in working with adult stem cells are that they have a finite life span and pluripotency can be lost. Microcarriers offer a large surface area for growth of anchorage-dependent cell types, and could thereby facilitate use of bioreactors for stem cell expansion in fewer passages. In this application note we expanded MSC cultures on Pall Solohill Collagen microcarriers (C102-1521) in spinner vessels and used cells from those spinner cultures to seed a three-liter disposable bioreactor (EMD Millipore ◆ ) containing two liters of medium. Maintenance of multipotency of MSCs expanded in bioreactor cultures was verified by immunostaining with stem cell-specific antibodies and by assessing their ability to differentiate into osteocytes and adipocytes in vitro.
2.1
Culturing MSCs
Population doubling level (PDL) 19 bone marrow-derived MSCs were donated from EMD
Millipore and expanded in Dulbecco’s Modified Eagle Medium, (DMEM; Gibco ◆ 11054) supplemented with 10% fetal bovine serum (FBS; Thermo Scientific HyClone SH30071.03),
2 mM L-glutamine (Thermo Scientific HyClone SH30034.02), penicillin/streptomycin
(ATCC 30-2300), and basic Fibroblast Growth Factor (bFGF; (EMD Millipore GF003).
For initial expansion on flatware, MSCs were cultured on Corning ◆ T-flasks (430825, 430639, and 430641). To subculture cells, medium was decanted and cells were rinsed once with
Dulbecco’s Phosphate Buffered Saline (DPBS, Thermo Scientific HyClone SH30028.03).
DPBS was immediately decanted and 1 to 3 mL of TrypLE ◆ Select (Gibco 12563) was added at a surface-to-volume ratio of 20 μL/cm 2 . Flasks were incubated at 37 °C until cells detached
(5 to 8 minutes). Cells were resuspended in medium and centrifuged at 300 g for five minutes to pellet cells. Medium and trypsin were decanted and cells were resuspended in 2 to 5 mL of complete medium without bFGF (volume was dependent on number and size of T-flasks harvested). Cell counts were then performed using trypan blue and a Nexcelom counter with associated software. Fresh T-flasks were seeded at 5 x 10 3 cells/cm 2 and bFGF was added
(8 ng/mL) to seeded T-flasks, which were then incubated at 37 ºC ± 0.5 with 5% CO
2
. www.pall.com/biopharm 3

4
2.2
2.3
Medium exchanges (100% with fresh bFGF) were performed every third day beginning on the third day of culture. To quantify cell numbers and determine viability on flatware and spinners, cell counts were performed using the trypan blue method and a hemocytometer. For spinner cultures, samples were retrieved daily and nuclei counts using the citric acid/crystal violet method were performed. Nuclei were counted using a Nexcelom counter with associated software and the number of nuclei per cm 2 surface area was calculated for each sample.
Growth in Spinner Cultures
SoloHill microcarriers were prepared according to manufacturer’s instructions by autoclaving at 121 ºC in deionized water. Spinner cultures were essentially performed as described in the microcarrier technical documentation 7 . For initial expansion on microcarriers, MSCs were trypsinized from T-150s, processed to create a seed stock and planted onto a total surface area of 2060 cm 2 of Collagen microcarriers at 5 x 10 3 cells/cm 2 (two x 200 mL spinners corresponding to a microcarrier concentration of 14 g/L in each spinner). Spinners were incubated at 37 ºC ± 0.5 ºC with 5% CO
2
. Initial attachment in spinner cultures was performed under low serum conditions for one hour. After attachment, FBS to a final concentration of
10% and bFGF to 8 ng/mL were added and spinners were grown at 40 rpm for seven days.
For trypsinization of cells from microcarriers (spinner cultures), cells/microcarriers were allowed to settle and the medium was carefully removed by pipetting. Cells and microcarriers were washed with DPBS for five minutes at room temperature with occasionally rocking back and forth by hand to resuspend microcarriers. After five minutes, microcarriers were allowed to settle, DPBS was removed and 10 mL of TrypLE Select was added. The spinners were gently pipetted twice to thoroughly mix and then incubated at 37 ºC for 10-15 minutes (with occasional rocking by hand). Cells and microcarriers were pipetted after five minutes and again after ten minutes to achieve a single-cell suspension. After achieving a single-cell suspension, the cell and microcarrier slurry was passed through a 70 micron strainer, which retained the microcarriers.
The strainer was then flushed with complete medium. The cells were then centrifuged and resuspended in complete medium and were used to seed bioreactors.
Expansion into bioreactors
For expansion into bioreactors, microcarriers were prepared in a separate, autoclavable bottles by adding 28.6 grams of C102-1521 plus 100 ml diH
2
O, and autoclaving at 121 °C for 30 minutes.
(5150 cm 2 /L x 2 L= 10300 cm 2 total, 10300 cm 2 ÷ 360 cm 2 /gram = 28.6 grams Collagen microcarrier). DO and pH probes were autoclaved separately. Once autoclaved, the microcarriers, pH and DO probes were added to the bioreactor aseptically in a biological safety hood. Medium without FBS was added to the bioreactor and temperature was stabilized at 37 °C using an electrical heating blanket.
After a stable environment was established, the bioreactor was seeded at 5 x 10 3 cells/cm 2 .
After one hour in low-FBS medium, attachment of cells was greater than 95%. FBS and bFGF were then added at final concentrations of 10% and 8 ng/mL respectively, and the culture was grown at 75 rpm. This agitation rate was determined empirically as the lowest rate required to keep the microcarriers suspended from the bottom of the vessel and fairly uniformly mixed throughout the entire bioreactor volume. After three hours, a sample was taken to verify attachment uniformity. The numbers of cells per microcarrier were counted for 100 microcarriers and plotted. Air overlay was sufficient for control of pH and oxygen overlay for percent dissolved oxygen. In the first 24 hours after cell addition the pH increased slightly (cells were added at pH 7.4) and 5% CO
2 was overlayed into the head space to maintain pH at 7.4.
On day four, a 75% volume batch medium exchange was performed. Cells and microcarriers were allowed to settle for 10 minutes, spent medium was removed via a side port on the bioreactor vessel; then fresh medium containing 8 ng/mL bFGF and 10% FBS was added to the vessel. Cells were cultured through day seven with samples retrieved daily to assess cell growth.

Cells within the bioreactor were harvested with trypsin and subsequently characterized. For trypsinization, microcarriers were allowed to settle and as much medium as possible was removed through a side port on the bioreactor vessel. One liter of sterile PBS was added to the vessel and the cell-laden microcarriers were washed for ten minutes at 37 °C at 75 rpm. After the PBS wash, the entire volume was transferred through the bottom drain port into a sterile one liter glass bottle for trypsinization in a biological hood. Once microcarriers settled and PBS was removed, 200 mL of TrypLE Select was added. Cells were incubated at 37 °C for 30 minutes with occasional swirling of the bottle to disrupt cells from microcarriers. After incubation at 37 °C, cells were passed through a sterile 100 micron mesh screen. Microcarriers were retained by the screen and cells readily passed through. Microcarriers retained on the screen were flushed with complete medium and a cell count was performed on the final cell suspension.
Expression of stem cell markers on MSCs expanded on SoloHill microcarriers was quantified by using flow cytometry. Trypsinized cells were transferred into a 50 mL tube, cells were centrifuged (300 g for five minutes) and resuspended in 4% paraformaldehyde for 15 minutes at room temperature. Paraformaldehyde was removed and cells were washed in DPBS and stored at 4 °C until flow cytometry analysis.
To determine differentiation potential, cells were passaged from the bioreactor onto flatware
(24 well plate) at 3x10 4 cells/cm 2 . Growth/expansion medium was removed and one mL of either osteogenesis induction medium (Millipore SCR028) or adipogenesis induction medium (Millipore
SCR020) was added. Induction and maintenance media were changed according to Millipore’s protocol for cell differentiation. Osteocyte differentiation was determined by Alazarin Red S staining and adipocyte differentiation was determined by Oil Red O staining (protocols with
Millipore kits).
3.1
Growth of cells in bioreactor
Cells were seeded into bioreactors in medium containing low concentrations of FBS (less than
0.5%) and no bFGF to increase attachment rates and enhance distribution of cells across the microcarrier population. The average number of attached cells per microcarrier in the bioreactor was 3.7 versus 3.1 in the spinner control, showing slightly better attachment uniformity in the bioreactor (Figure 1), as 100% attachment and uniformity would yield ~3.9 cells/bead.
Approximately 6% of microcarriers were unoccupied in the bioreactor and 8% in the spinner at the three hour time point.
Figure 1
Cellspermicrocarrier
Asamplewastakenfromthebioreactorafter3hours,fixedandthenumberofcellsattachedtoindividualmicrocarriers wascountedfor100microcarriers.
www.pall.com/biopharm 5

The growth curve in Figure 2 shows that the MSC culture grew from 0.5 x 10 4 cells/cm 2 to just over 12 x 10 4 cells/mL in six days. This equates to a 32 hour doubling time, which is slightly faster than observed in flatware cultures (~48 hours). Similar results have been observed with microcarrier cultures previously.
Figure 2
Growthcurvesfrombioreactorandspinnercontrolcultures.
A drop in nuclei counts was accompanied by a slight increase in pH on day 7 (shown in
Figure 3). This could indicate that confluency is reached and signal a need for expansion.
Figure 3
Trendlinesforthecontrollersduringabioreactorrun.
6
Dissolvedoxygenisshowninblue.pHisshowninred.Temperatureisshowningreen,andtheagitationrateisshown inpink.
As shown in Figure 3, the demand for oxygen (DO) was low for the first 72 hours of culture, possibly due to the low number of total cells from the low seeding density. However, ten hours prior to the batch medium exchange, the DO dipped below 30%. Additionally, the batch medium exchange immediately restored the DO to ~85%, which subsequently started dropping.

The pH in the bioreactor initially climbed after cells were added, and as a response, 5% CO
2 was overlayed. This was performed three times over the first 48 hours until the pH stabilized.
After two days of culture, the pH was stable and slowly began decreasing until the batch medium exchange. Similar to the oxygen trend, the pH initially rose with the medium exchange but subsequently decreased and on day five, the pH value dipped just below 7.18, the set point for air overlay. Additionally, the pH began to climb towards the end of day seven. Taken together with the nuclei density decrease on day seven, pH could be used as an indicator of confluence and need for passage.
Figure 4 shows representative images of cells on microcarriers in samples retrieved from the bioreactor and control spinners on days five and seven. MSC-laden microcarriers began to aggregate earlier in the bioreactors whereas less aggregation was observed in spinner cultures.
Nuclei counts shown in Figure 2 support this visual observation of the slightly faster growth rate observed under bioreactor conditions. By day seven, aggregation was significantly more noticeable in the bioreactor sample, also shown in Figure 4.
Figure 4
ImagesofgrowthofMSCsoncollagenmicrocarriersina3-literbioreactorcomparedtogrowth inaspinnercontrol.
A)ImagesfromDays5and7duringthebioreactorgrowthshowinitialclumpingonday5andmoresignificantclumping onDay7.B)Imagesfromspinnergrowthshowsimilarresults.However,clumpingwasmoresignificantinthebioreactors.
After seven days of growth in the bioreactor, MSCs were trypsinized to determine viability, recovery, differentiation status and pluripotency. After an initial PBS wash (Figure 5A), cells appeared healthy and remained bound to microcarriers following this, a single-cell suspension was obtained through trypsinization (Figure 5B). After filtration through a 100 micron mesh screen,
495 million cells were recovered from the bioreactor.
www.pall.com/biopharm 7

Figure 5
MSCstrypsinizedfromCollagenmicrocarriersinthe3-literbioreactor.
8
A)After1literPBSwash,cellsremainattachedtothemicrocarriers,andappearhealthy.B)Priortofiltration,cellsare single-celledandcompletelydetachedfrommicrocarriers.
MSCs harvested from the bioreactors were either plated for differentiation or fixed and tested for stem cell markers to assess cell marker expression patterns. Figure 6 shows that cells trypsinized from the bioreactor and plated on flatware remain undifferentiated (Figure 6A) and are able to differentiate into adipocytes (B) and osteocytes (C) over 14 to 21 days, demonstrating that MSCs expanded on microcarriers retain pluripotency.
In addition to post-bioreactor differentiation, cells were formaldehyde-fixed, cells were formaldehyde fixed for determination of stem cell markers by flow cytometry. Greater than 90% of cells trypsinized from the bioreactor microcarriers retained expression of CD105, CD90 and CD44 relative to MSCs grown on t-flasks for flow cytometry control. As expected, cells were negative for expression of CD106, CD274, CD19, CD34, CD11b, CD79A, CD45, and CD14. The flow cytometry data validates the morphology of undifferentiated cells plated on flatware (Figure 6A) and shows that MSCs expanded on microcarriers can be recovered and maintain their undifferentiated phenotype.
Figure 6
MSCsgrownonSoloHillCollagenmicrocarriers.
MSCsinthebioreactorremainundifferentiatedwhileretainingpotentialtodifferentiateintoadipocytesandosteocytes.
Figure 7
FlowcytometrywasusedtodeterminepercentofcellsexpressingMSCmarkers.
100
Collagen Bioreactor
80 T Flask Control
60
40
20
0
CD
10
5
CD
90
CD
44
CD
10
6
CD
27
4
CD
19
CD
34
CD
11 b
CD
79
A
CD
45
CD
14
Marker
Greaterthan90%ofcellsexpressedCD105,CD90,andCD44cellmarkerstypicallyexpressedinundifferentiatedMSC populations.Andasexpected,lessthan10%ofcellsexpressedmarkersCD106,CD274,CD19,CD34,CD11b,
CD79A,CD45,andCD14.

Current obstacles limiting the use of stem cells for therapeutic benefits include a limited number of cell divisions and the potential loss of pluripotency. Due to the restricted number of population doublings, achieving maximal possible expansion in the fewest passages is vital. We have shown here that undifferentiated MSCs can be expanded to large numbers in a small volume bioreactor on microcarriers, while maintaining cell marker expression patterns and not affecting the ability of MSCs to differentiate into adipocytes and osteocytes. Considering the recoverable cell numbers presented here, a 4 L bioreactor volume would result in enough cells for a single therapeutic dose of ~1 x 10 9 cells. Ongoing studies are aimed toward optimizing growth and expansion of stem cells under animal component-free conditions.
1. Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. ExpHematol 1976, 4: 267-274.
2. Fraser etal.
Fat tissue: an underappreciated source of stem cells for biotechnology. TrendsBiotechnol
2006, 24: 150-154.
3. Cao C, Dong Y. Study on culture and in vitro osteogenesis of blood-derived human mesenchymal stem cells. ZhongguoXiuFuChongJianWaiKeZaZhi.
2005. 19: 642-647.
4. Griffiths Mu, Bonnet D, Janes SM. Stem Cells of the Aveolar epithelium. Lancet.
2005, 366: 249-260.
5. Beltrami etal.
Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell.
2003, 114: 763-776.
6. Pittenger etal.
Multilineage potential of adult human mesenchymal stem cells. Science.
1999, 284:
143-147.
Visit us on the Web at www.pall.com/biopharm
E-mail us at microcarriers@pall.com
Customer Ordering
Pall Life Sciences
4370 Varsity Drive, Suite B
Ann Arbor, MI 48108 microcarriers@pall.com
734-973-2956 phone
European Headquarters
Fribourg, Switzerland
+41 (0)26 350 53 00 phone
LifeSciences.EU@pall.com e-mail
Corporate Headquarters
Port Washington, NY, USA
+1.800.717.7255 toll free (USA)
+1.516.484.5400 phone biopharm@pall.com e-mail
Asia-Pacific Headquarters
Singapore
+65 6389 6500 phone sgcustomerservice@pall.com e-mail
International Offices
Pall Corporation has offices and plants throughout the world in locations such as: Argentina,
Australia, Austria, Belgium, Brazil, Canada, China, France, Germany, India, Indonesia, Ireland,
Italy, Japan, Korea, Malaysia, Mexico, the Netherlands, New Zealand, Norway, Poland, Puerto
Rico, Russia, ingapore, South Africa, Spain, Sweden, Switzerland, Taiwan, Thailand, the United
Kingdom, the United States, and Venezuela. Distributors in all major industrial areas of the world. To locate the Pall office or distributor nearest you, visit www.pall.com/contact
The information provided in this literature was reviewed for accuracy at the time of publication.
Product data may be subject to change without notice. For current information consult your local Pall distributor or contact Pall directly.
© 2015, Pall Corporation. Pall, , Hillex and SoloHill are trademarks of Pall
Corporation. ® indicates a trademark registered in the USA and TM indicates a common law trademark. Filtration.Separation.Solution.
is a service mark of Pall Corporation.
◆ Corning is a trademark of Corning, Inc.; Gibco is a registered Trademark of Life
Techologies, Inc.; TrypLE is a trademark of Life Technologies, Inc. and EMD Millipore is a registered trademark of KGaA
2/15, PDF, GN15.6127
USD2978 (2)