Materials Science & Engineering C 96 (2019) 77–85

Contents lists available at ScienceDirect

Materials Science & Engineering C

journal homepage: www.elsevier.com/locate/msec

Evaluation of reagents used to coat the hollow-fiber bioreactor membrane of T

the Quantum® Cell Expansion System for the culture of human mesenchymal
stem cells☆
Nathan D. Frank , Mark E. Jones, Boah Vang, Claire Coeshott
⁎

Terumo BCT®, Inc., Lakewood, CO, USA

ARTICLE INFO ABSTRACT

Keywords: The addition of a coating reagent to promote cell adherence is necessary to prepare the membrane surface of the
Mesenchymal stromal cell Quantum® Cell Expansion System hollow-fiber bioreactor for the culture of mesenchymal stem cells. In this
Hollow-fiber bioreactor study, the efficacy of 8 potential coating reagents has been compared in terms of the doubling times of their cell
Regenerative medicine populations, cell morphology, characterization via flow cytometry, and capacity for trilineage differentiation.
Cell therapy
Human fibronectin (FN), pooled human cryoprecipitate (CPPT), and recombinant human vitronectin (VN) were
Extracellular matrix
Automated cell culture
successful as coating reagents, and each product has advantages in different cell culture contexts. Mesenchymal
stem cells harvested from Quantum cultured with each of these 3 compounds as coating reagents all met
International Society for Cellular Therapy standards for plastic adherence, surface marker expression, and
successful trilineage differentiation. No significant differences were observed among the doubling times from
Quantum harvests using FN, CPPT, or VN as coating reagents (P = 0.31). Coating with gelatin, human serum
albumin, collagen I, poly‑L‑lysine, and poly‑D‑lysine resulted in significantly lower harvest yield; these agents are
not recommended for use as coating reagents in the Quantum system.

1. Introduction therapy space.

The number of clinical trials investigating the use of mesenchymal
stem cells (MSCs, also known as mesenchymal stromal cells or medic-
inal signaling cells) as therapy for a range of indications increased from
206 to 493 between the years of 2012 and 2015 [1]. Patients suffering
from autoimmune disorders such as Crohn's disease [2] and systemic
lupus erythematosus [3], as well as those requiring regenerative
therapies for tissue repair [4], have been shown to benefit from MSC
treatments. Clinical trials are currently ongoing to investigate MSCs as
potentially therapeutic in a range of indications from graft-versus-host
disease to traumatic brain injury [5,6]. As interest in producing MSC-
based therapies continues to grow, the need for reliable methods of
consistent, larger-scale MSC expansion for the therapeutic market is
growing apace. Flask-based MSC culture is time-consuming and cum-
bersome. Furthermore, the use of flask cultures to expand enough MSCs
to achieve the estimated clinical doses of 1 to 5 × 106 MSCs/kg body
weight required for many MSC-based therapies [7] is inherently risky
due to the number of repeated manipulations and open events required.
A more reliable, consistent process for MSC culture is needed in the cell
The Quantum® Cell Expansion System (Quantum system; Terumo
BCT, Inc., Lakewood, CO) is a functionally closed, hollow-fiber bior-
eactor (HFB) system designed to culture both adherent [8,9] and sus-
pension cells (Fig. 1). The HFB of the Quantum system comprises
~11,500 polyether sulfone fibers totaling 2.1 m2 of available surface
area for the culture of adherent cell types; this is approximately
equivalent to the surface area of 120 T175 tissue culture flasks.
The HFB itself is fully integrated into the disposable set (Fig. 2) and
is divided into separate intracapillary (IC) and extracapillary (EC) fluid
pathways. The fluidics for the IC and EC pathways are maintained by
inlet pumps that determine the flow of new medium into each side of
the bioreactor, and circulation pumps that determine the rate at which
the medium in each side of the bioreactor is moved through its circuit
(Fig. 3). Users have the option to set the IC and EC sides to be open to
one another or to limit communication between the two sides to what is
filtered through the HFB membrane. The Quantum system's ability to
reproducibly culture large numbers of expanded MSCs from whole bone
marrow (WBM) in less time and with fewer doublings than manual T-
flask culture has been demonstrated [10], and the genetic stability of

☆
Funding: all funding for this work was provided by Terumo BCT®, Inc.
⁎
Corresponding author at: 10810 W. Collins Ave., Lakewood, CO 80215, USA.
E-mail address: Nathan.Frank@TerumoBCT.com (N.D. Frank).

https://doi.org/10.1016/j.msec.2018.10.081
Received 29 March 2018; Received in revised form 28 September 2018; Accepted 23 October 2018
Available online 26 October 2018
0928-4931/ © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
N.D. Frank et al.
Fig. 1. Image of the Quantum system. Left: system with disposable set and HFB attached. Right: system with active graphic user interface and media/waste bags in
use.
Fig. 2. Quantum disposable set featuring integrated harvest bag, waste bag, tubing organizer, and HFB.
the MSCs produced using Quantum has been confirmed [11].
In its untreated condition, the inner aspect of the IC side of the
bioreactor is ready to accept an inoculum of suspension cells. However,
to prepare the bioreactor for the culture of an adherent cell type like
MSCs, it is recommended that the user add an adherence-promoting
compound to the IC loop of the bioreactor to facilitate cell attachment
and proliferation. Since the system's commercial release in 2011, fi-
bronectin (FN) from human plasma has been the standard coating re-
agent used in the Quantum system. However, because a single product
may not suit the needs of all users, a variety of coating agents have been
explored to provide alternative options and to and to demonstrate
which compounds may not be effective for HFB coating in preparation
for seeding with adherent cells.
Extracellular matrix (ECM) proteins are a fixture of the MSC mi-
croenvironment. The external cues provided by contact with the ECM
lead to changes that can affect cell shape and motility, as well as acting
to initiate signaling cascades that can affect cell differentiation and
proliferation [12]. Integrin proteins on the cellular surface act as both
sensors and effectors. They read the forces and molecular signals pro-
vided by the matrix to which cells adhere and translate them into the
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Materials Science & Engineering C 96 (2019) 77–85
physical actions required for cell motility, replication, and spreading
[13]. The molecular composition of the cell culture substrate is also
highly relevant to cell behavior. Work conducted by Song et al. [14]
showed that MSCs cultured on silicone surfaces coated with type 1
collagen displayed a dose-dependent reduction in proliferative poten-
tial, while those same surfaces coated with human FN did not enhance
or inhibit MSC proliferation at any dose when compared to uncoated
tissue culture plastic (TCP) [15].
In this study, potential coating reagents were investigated for their
ability to support the attachment and expansion of MSCs in Quantum.
Three coating reagents (FN from human plasma, recombinant VN, and
human CPPT) were found to be consistently effective, and Quantum
users can choose from these options when deciding how best to tailor
their MSC microenvironment through the application of ECM proteins
to which MSCs attach in their native niche. Previously, MSC attachment
and expansion in TCP flasks has been compared to that achieved when
seeding bone-marrow-derived MSCs in the Quantum HFB coated with
FN [10] and when seeding adipose-derived MSCs in the Quantum HFB
coated with CPPT [16]. Quantum MSC harvests have also yielded fa-
vorable results when compared to multilayer cell stacks in terms of
N.D. Frank et al.
Fig. 3. Disposable set attached to the Quantum system. Image features intracapillary/extracapillary (IC/EC) inlet pumps, circulation pumps, integrated gas transfer
module, and gas inlet.
their yield and overall cost to manufacture a given number of MSCs
[17]. For these reasons, MSC adherence to TCP was not included in this
comparison. Rather, the goals of this work were 1) to evaluate a variety
of potentially effective coating reagents for the Quantum system and
separate those that were successful from those that were not, and 2) to
demonstrate certain other compounds were not effective as coating
reagents and should be avoided when a Quantum user is considering
candidates for possible coating reagents.
The MSCs expanded for this work were limited to the P0 to P2
passages as these are the passages thought to be most therapeutically
desirable in the clinic. For example, first- or second-passage MSCs have
been shown to be notably more effective at prolonging patient survival
in cases of graft-versus-host disease (GVHD) when compared to third- or
fourth-passage MSCs [18]. Other work has demonstrated that MSCs
begin to enter cellular senescence from the moment they are removed
from their physiological niche and placed into an ex vivo culture en-
vironment. Over time, these MSCs exhibited shortened telomere length,
a reduction in proliferative potential, and reduced differentiation ca-
pacity [19,20]. As such, earlier passages of MSCs were evaluated for
this study.
In this study, we have identified three coating reagents for MSC
culture in the Quantum system that generated equivalent cell products
in terms of cell doubling time, post-harvest adherence to TCP, cell
morphology, surface marker expression, and trilineage differentiation.
MSCs harvested from Quantum runs using gelatin, human serum al-
bumin (HSA), collagen I, poly‑L‑lysine, and poly‑D‑lysine as coating
reagents were compared only in terms of their harvest yield relative to
FN.
2. Materials and methods
2.1. Materials
Healthy, fresh, adult human whole bone marrow (WBM) was pur-
chased either from AllCells LLC (Alameda, CA) or from Lonza (Basel,
Switzerland). Flow cytometry staining buffer was purchased from BD
Biosciences (San Jose, CA). FN from human plasma (5 mg) was pur-
chased from Corning Inc. (Corning, NY). CPPT was purchased from
Bonfils Blood Center (Denver, CO). Cellgro gelatin solution (0.1%) was
purchased from CellGenix Inc. (Portsmouth, NH). Gelatin solution
(2%), poly‑L‑lysine, poly‑D‑lysine, hematoxylin solution, formalin, and
collagen solution were purchased from Sigma-Aldrich (St. Louis, MO).
Human serum albumin was from Invitria (Fort Collins, CO).
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Materials Science & Engineering C 96 (2019) 77–85
Recombinant human vitronectin (VN), αMEM + GlutaMAX, fetal bo-
vine serum (FBS), trypsin 0.25% with EDTA, and StemPro® adipogenic,
osteogenic, and chondrogenic differentiation kits were purchased from
Thermo Fisher (Waltham, MA). Phosphate buffered saline (PBS) was
purchased from Lonza Group (Basel, Switzerland). Alcian blue, alizarin
red S sodium salt, and oil red o isopropanol solution were purchased
from VWR (Radnor, PA).
2.2. Equipment
The Quantum system, Quantum cell expansion disposable sets,
Quantum 1-L cell inlet bags, Quantum 4-L media bags, Quantum 4-L
accessory bags, Quantum 200-μm in-line filters, and the TSCD®-Q
Sterile Tubing Welder were manufactured by Terumo BCT (Lakewood,
CO). Peristaltic pumps were purchased from Masterflex (Vernon Hills,
IL). i-STAT Portable Clinical Analyzer, i-STAT G cartridges, and i-STAT
CG4+ cartridges were purchased from Henry Schein (Melville, NY).
MSCs were counted using a Vi-CELL XR Cell Counter from Beckman
Coulter (Fort Collins, CO).
2.3. Reagent preparation and coating of bioreactor
All coating reagents were prepared according to manufacturer re-
commendations, brought up to a total of 100 mL in PBS, and passed
through a 0.22-μm Quantum filter accessory prior to being loaded into
cell inlet bags. Cell inlet bags containing each coating reagent were then
attached to the Quantum system and the coating process was initiated.
One hundred milliliters of each coating reagent were pumped into the
IC loop of the bioreactor, which has a total circulating volume of
189 mL. Concentrations in terms of this final dilution into 189 mL of
PBS are, for FN: 5 mg, 60 nM; VN: 4 mg, 282 nM; human collagen I
solution: 6 mg, 235 nM; poly‑L‑lysine/poly‑D‑lysine: 10 mg, 0.10 g/L;
human serum albumin: 1 g, 79.6 μM.
Pooled CPPT products used for these studies contained processed
plasma from 5 donors. Protein concentrations were not determined.
CPPT was stored at −20 °C until use, and the thawed product was di-
luted to 500 mL with PBS. This stock solution was aliquoted and stored
again at −20 °C. Cryoprecipitate demonstrated no decline in function
as a coating reagent after 3 freeze/thaw events (unpublished results).
To prepare CPPT solution for bioreactor coating, one 25 mL aliquot was
mixed with 75 mL PBS.
To maintain a functionally closed environment during cell culture
media preparation, a 4-L media bag kit was used that included an inline
N.D. Frank et al.
0.22-μm filter pod through which the medium passed as it was pumped
into the media bag. The media bag was then attached to the appropriate
inlet line on the Quantum disposable set. Complete medium, α-
MEM + GlutaMAX supplemented with 10% FBS, was used throughout
these studies.
2.4. Preparation for MSC culture in Quantum
To maintain a reliably closed environment for every cell culture
event, Quantum makes use of a disposable cassette, which includes an
integrated 2.1-m2 HFB, a 1-L harvest bag for cell collection, and a 4-L
accessory bag to collect used PBS or other spent media products that
accumulate as waste during the culture process. The disposable set and
its associated HFB are contained within an incubation chamber that is
set to maintain a constant 37 °C environment.
Manipulations of the system's fluidics and other tasks are accom-
plished using a touch-screen user interface. The disposable sets used
with Quantum each contain an integrated gas transfer module that can
be connected to an outside source to provide the gas composition of the
user's choosing. For all experiments described here, tanks containing
20% O2, 5% CO2, and 75% N2 were used to culture MSCs. All MSC
cultures described herein were expanded on the IC side of the Quantum
HFB while maintaining a circulation rate of 30 mL/min on the EC side
to provide continuous gas exchange and waste removal across the
membrane. Prior to HFB coating, the system was primed with ap-
proximately 1.5 L PBS.
2.5. Overnight bioreactor coating
After the disposable set was primed with PBS, a cell inlet bag con-
taining the coating agent was sterile-welded to the appropriate inlet
line. The integrated Coat Bioreactor task was used to draw the coating
solution into the IC loop and to circulate the solution throughout the IC
loop for 16 to 24 h before the solution was replaced with complete
medium and the cell inoculum was introduced to the HFB. Coating of
the HFB occurred inside Quantum's incubator chamber at 37 °C.
2.6. MSC culture in Quantum
2.6.1. WBM passage: P0
Prior to a WBM load, the bioreactor was coated overnight. WBM
cultures were expanded using 1 of only 3 coating types—FN, CPPT, or
VN—because the doubling times (dTs) from Quantum systems using the
other coating agents tested (gelatin, human serum albumin, collagen I,
poly‑L‑lysine, and poly‑D‑lysine) were deemed too high to warrant a
WBM expansion (Table 1). WBM (12.5–25 mL/Quantum) was diluted to
100 mL in complete medium. WBM solution was loaded into the IC loop
and no circulation was imposed for 24 h to allow MSC attachment to the
coated inner surface of the HFB membrane. After 24 h, the IC loop was
washed with complete medium to gently remove nonadherent cell types
while leaving behind adherent cells anchored to the bioreactor
Table 1
MSCs were cultured in Quantum following overnight coating with each reagent
listed. Percent values are given as relative to the harvest value from a control
Quantum system coated with human FN.
Coating reagent % of FN-coated control HFB harvest yield
5 mg FN (control) 100
100 mL gelatin 0.1% 12
100 mL gelatin 2.0% 10
189 mL gelatin 2.0% 9 via flow cytometry. CD73 and CD90 were conjugated to phycoerythrin
6 mg collagen 1 8 (PE). CD105, CD34, and CD19 were conjugated to allophycocyanin
1 g human serum albumin 7
10 mg poly‑L‑lysine 8
10 mg poly‑D‑lysine 9 isothiocyanate (FITC). CD146 antibody was conjugated to a PE-Cy7
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Materials Science & Engineering C 96 (2019) 77–85
membrane.
As MSCs make up only 0.001% to 0.01% of the total population of
mononuclear cells in a WBM sample [14], a relatively low IC inlet rate
of 0.1 mL/min was typically sufficient for an entire WBM expansion
lasting from 9.7 to 13.8 days. When feed rate did require adjustment, a
30% drop in glucose values relative to the baseline was used as an in-
dicator to double the feed rate of the system to accommodate a dou-
bling population of cells.
To harvest MSCs from Quantum, 180 mL of trypsin was placed into
an inlet bag and sterile-welded to the appropriate inlet line. Integrated
system tasks were used to wash the MSCs with PBS, pump the trypsin
into the HFB, and flush the harvest product into the attached harvest
bag using complete medium to neutralize the activity of the trypsin.
2.6.2. Preselected MSC passages: P1 and P2
HFBs were coated overnight with the appropriate coating agent
prior to loading MSCs. Between 164 MSCs/cm2 and 500 MSCs/cm2
were loaded into the bioreactor depending on the requirements for the
experiment set in question. MSCs were given 24 h without IC loop
circulation to attach to the coated membrane surface. Cell populations
were expanded for 4.8 to 7.0 days.
MSC feeding method varied by experiment type. MSCs were cul-
tured either using a reactive feeding strategy such as that described in
the P0 MSC Feeding section above or using a scheduled feeding strategy
in which feed rates were adjusted upward on a set schedule rather than
in response to a metabolite reading. The procedures for an MSC harvest
from P1 and P2 passages were identical to those described in the P0
MSC Harvest section above. Daily readings of glucose and lactate values
were taken by drawing a 1 mL sample of cell supernatant through the
EC-sample port and were read using the i-STAT analyzer (data not
shown)
2.6.3. MSC doubling time calculations
MSC dT was used as a comparative metric for this analysis, as the
equation accounts for differences in the number of MSCs seeded, the
number of MSCs harvested, and the culture duration. Cell dTs were
calculated using the following equation [21]:
ln 2(culture time in hours)
Cell dT =
ln ( MSCs harvested
MSCs inoculated )
2.6.4. MSC assays
2.6.4.1. Flow cytometry. Cells (1.5 × 106) were incubated in a cocktail
of antibodies used at manufacturers' recommended concentrations and
washed in stain buffer, and data were acquired on a FACSCanto II flow
cytometer using FACSDiva software (BD Biosciences, San Jose, CA).
Appropriate isotype controls were included to facilitate population
gating. Ten thousand events per tube were acquired. Harvested MSCs'
potential for trilineage differentiation was measured by calculating the
median fluorescence intensity ratio for the CD146+ MSCs. Median
fluorescence intensity ratio was calculated by dividing the CD146+
population median fluorescence intensity by the unstained population
median fluorescence intensity; a value > 10 indicates that the MSC
population is capable of differentiation into adipogenic, osteogenic, and
chondrogenic lineages [22]. Antibodies for MSC characterization were
specific for CD73 (clone AD2), CD14 (M5E2), CD19 (HIB19), CD34
(581), CD45 (HI30), HLA-DR (Tu39) (all from BD Biosciences); CD146
(SHM-57) (BioLegend, San Diego, CA); and CD90 (eBio5E10) and
CD105 (SN6) (Thermo Fisher). Each antibody was tagged with a
fluorescent labeling molecule to facilitate visualization and analysis
(APC). CD14, CD45, and HLA-DR were tagged with fluorescein
tandem dye.
N.D. Frank et al.
2.6.4.2. Cell morphology. MSCs from each Quantum harvest were
seeded in 24-well plates at 1.0 × 104/well. Plates were incubated at
37 °C, 5% CO2. Cells were evaluated microscopically and 100× images
were taken between days 1 and 3 to confirm appropriate MSC
adherence to plastic, as well as typical MSC morphology.
2.6.4.3. Trilineage differentiation. Trilineage differentiation was
performed on select Quantum system harvest products: P2 harvest
products from the FN- and VN-coated units and P1 harvest products
from the CPPT-coated units. Adipogenic, osteogenic, and chondrogenic
differentiation were performed per the vendor's instructions. Staining of
adipocytes was performed using oil red o, chondrocytes were stained
using alcian blue, and calcium deposits indicative of osteogenesis were
stained with alizarin red. Differences in the relative amounts of
differentiation among MSCs cultured on different substrates were not
compared, and assessment of differentiation cultures was done on a
pass/fail basis with the presence of stained adipocytes, stained calcium
deposits, and stained chondrocytes defined as positive results for
adipogenesis, osteogenesis, and chondrogenesis, respectively.
2.6.4.4. Statistical analysis. Statistical analysis of data was performed
using GraphPad Prism 7. A Kruskal-Wallace one-way ANOVA was used
to detect significance.
3. Results
3.1. Doubling times: FN, CPPT, and VN
There were no significant differences in dTs observed among
Quantum harvests using FN, CPPT, or VN as coating reagents
(P = 0.31) (Fig. 4). Doubling times for P0 passages were not included in
this analysis, as WBM expansions contained a mixed cell population and
estimates of colony-forming unit counts for each culture were not
considered sufficiently robust to determine population dTs for those
runs (data not shown). P1 and P2 dTs were aggregated into a P1 and P2
group that excluded values from P0 harvests to facilitate analysis.
Comparative dTs of P1 and P2 MSCs harvested from Quantum using
5 mg FN, 25 mL CPPT solution, or 4 mg VN as coating agents. Each box
represents the median 50% of dTs for the coating reagent in question;
whiskers represent the 25% highest and 25% lowest dTs for cultures.
Two outlier dTs represent results from the P1 (40.2 h) and P2 (41.6 h)
passages from a single donor's MSCs cultured using CPPT as a coating
agent.
Fig. 4. P1 and P2 dTs of MSCs cultured using FN, CPPT, or VN as coating re-
agents.
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Materials Science & Engineering C 96 (2019) 77–85
3.2. Harvest yield: other coating reagents
Quantum harvests of MSC yields from HFBs coated with FN were
compared to Quantum harvests of MSC yields from HFBs coated with
one of these: 0.1% gelatin, 2.0% gelatin (100 mL and 189 mL), collagen,
HSA, poly‑L‑lysine, or poly‑D‑lysine. Harvest yield for all conditions fell
between 7% and 12% of the FN-coated control (Table 1).
3.3. Harvest yields
Of 18 Quantum MSC expansions in which FN coating was used, 2
were P0 (WBM) passages, 7 were P1 passages, and 9 were P2 passages.
FN-cultured P0 harvests had MSC yields of 1.7 × 108 and 5.5 × 107.
For FN-cultured P1 and P2 harvests, MSC yield ranged from 5.6 × 107
to 5.4 × 108. Of 21 Quantum MSC harvests that used CPPT as a coating
reagent, 3 were P0, 6 were P1, and 13 were P2 passages. P0 harvest
yields were 1.3 × 108, 1.1 × 108, and 5.3 × 107. For CPPT-cultured P1
and P2 harvests, MSC yield ranged from 1.1 × 108 to 5.6 × 108. One
MSC harvest using VN as a coating agent was a P1 expansion; the re-
maining 6 were P2 expansions. VN-cultured P1 and P2 harvests ranged
from 6.4 × 107 to 4.7 × 108. These Quantum runs were conducted
using a variety of experimental conditions and achievement of higher
MSC yields were not necessarily the goal of each set of runs.
3.4. MSC morphology
Images were captured of MSCs from representative harvests to
confirm that MSC morphology was not affected by different coating
reagents. All MSCs displayed the expected adherence to plastic as well
as spindle-shaped morphology in post-harvest culture regardless of the
coating reagent used to culture the MSCs (Fig. 5).
3.5. MSC phenotype
MSCs harvested from Quantum using FN, CPPT, and VN were
evaluated for surface marker expression according to the International
Society for Cellular Therapy (ISCT) standards defined in Dominici et al.
[23]. The ISCT MSC flow cytometric criteria specify that positive
marker (CD73, CD90, CD105) expression by the population shall
be > 95%, and negative marker expression (CD14, CD19, CD34, CD45,
HLA-DR) shall be < 2%. All P1/P2 MSCs evaluated met these standards
regardless of the coating reagent used (Table 2). CD146+ expression
ratios must be > 10 relative to the unstained population to indicate that
the MSC population is capable of trilineage differentiation. Positivity
for CD146 expression is associated with a higher degree of multi-
potency in MSCs [24]. Cells expanded on FN, CPPT, and VN for these
experiments all had CD146+ expression ratios that exceeded the
minimum standard of > 10 times the unstained values (Table 2).
3.6. Trilineage differentiation
Trilineage differentiation was performed on representative MSC
harvests from Quantum HFBs that were coated with FN, VN, or CPPT.
Cells cultured using these 3 coating reagents all retained the ability to
differentiate into adipocytes, chondrocytes, or osteocytes (Figs. 6, 7,
and 8).
4. Discussion
The role of the surface to which MSCs attach is not limited to that of
an inert scaffold. Rather, the substrate on which MSCs are cultured has
the potential for both profound and subtle influence over the way that
the cells will behave in future culture and as therapeutic products. The
protein composition and physical properties inherent in the type of
substrate on which cells attach and proliferate are critical factors to
consider when culturing MSCs in a manufactured cell niche. For
N.D. Frank et al.
A B
C
Fig. 5. Representative images of MSC morphology (100×). A) MSCs cultured in Quantum using FN as a coating reagent. B) MSCs cultured in Quantum using CPPT as
a coating reagent. C) MSCs cultured in Quantum using VN as a coating reagent.
Table 2
Flow cytometry for MSCs harvested from Quantum HFB coated with FN, CPPT,
or VN.
HFB coating P1 and P2 mean CD146+ Do P1 and P2 MSCs meet
expression ratio ISCT minimal MSC criteria?
Fibronectin (n = 5) 164.9 Yes
Cryoprecipitate (n = 6) 175.6 Yes
Vitronectin (n = 3) 329.0 Yes
instance, adherence to a particular ECM molecule may predispose a cell
population toward differentiation in a particular lineage: MSCs cultured
on surfaces coated with VN and collagen have shown a tendency toward
increased osteogenesis, but this same effect is not seen in MSCs cultured
on FN-coated surfaces [25,26]. In another study, MSCs expanded on
ECM-coated scaffolds displayed increased metabolic activity and de-
creased apoptosis, and they secreted more proangiogenic factors than
on non-ECM-coated scaffolds [27]. Thus, expanding MSCs on a certain
substrate can be used as a deliberate method to influence a specific
differentiation pathway without the introduction of additional exo-
genous induction factors.
In addition to the composition of a substrate, the relative elasticity
of a substrate contributes to determining MSC fate as well. In the pre-
sence of no added growth factors, MSCs can be guided toward the
chondrogenic and osteogenic lineages by culturing them on more
elastic surfaces relative to TCP [28]. The decision of which coating to
use in a cell expansion process is an influential one and preparing the
Quantum HFB for MSC seeding is an opportunity for the user to define
the cellular microenvironment to suit the needs of a particular process
or product.
This study evaluated 8 compounds as potential coating reagents for
the HFB of the Quantum system to support the growth and expansion of
MSCs. Human FN has been the most frequently used and thoroughly
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Materials Science & Engineering C 96 (2019) 77–85
evaluated coating reagent for the Quantum HFB. FN is a defined, single-
protein product sourced from human plasma. Using 5 mg of FN to coat
the 21,000 cm2 of the IC side of the HFB has been demonstrated to be
effective for MSC seeding in the HFB. However, the manufacturer re-
commends using > 20 mg of FN to coat a similar surface area (FN
product insert). This discrepancy may speak to the efficiency of the
coating process in the Quantum HFB relative to that used to coat sur-
faces such as those in a tissue culture flask.
Cryoprecipitate is also a well-tested coating reagent for use in the
Quantum HFB, and CPPT has previously been used effectively as a
coating for tissue culture flasks [29]. CPPT is produced by blood centers
to meet minimum standards for fibrinogen (≥150 mg/unit) and factor
VIII (≥80 IU/unit) [30], but the concentrations of FN, VN, and other
plasma glycoproteins are typically not measured upon production. As
such, the efficacy of CPPT as a HFB coating reagent may vary between
manufacturers. Callum et al. [31] measured the FN level in CPPT at
1.5 mg/mL. This would indicate that, at the concentrations used in
these studies for coating a HFB, ~7.5 mg FN would be present in the
coating solution along with fibrinogen, VN, and other substances. This
lack of definition makes CPPT a unique substance to use for the culture
of MSC therapeutics, as it is not rated as a good manufacturing practice
(GMP)-grade product, but it is produced as a biologic intended for di-
rect introduction into human patients.
The VN used for these studies was produced by recombinant tech-
nology and is free of animal proteins. In addition to its being a re-
combinant product, this VN is manufactured in both research-grade and
GMP-grade versions (CTS A27940 VN). While GMP-grade VN was not
evaluated for this study, it would be advantageous for users who aim to
transition from research to clinical evaluation of an expanded MSC
product to make use of a research-grade coating reagent that would
essentially be the same product other than the increased testing per-
formed on the GMP version.
N.D. Frank et al. Materials Science & Engineering C 96 (2019) 77–85

A B

C D
Fig. 6. Adipogenesis (100×). A) Uninduced MSCs (cultured in Quantum using FN as a coating reagent). B) Induced MSCs (cultured in Quantum using FN as a coating
reagent. C) Induced MSCs (cultured in Quantum using CPPT as a coating reagent). D) Induced MSCs (cultured in Quantum using VN as a coating reagent). Positive oil
red o staining indicates the presence of differentiated adipocytes.

A B

C D
Fig. 7. Osteogenesis (400×). A) Uninduced MSCs (cultured in Quantum using FN as a coating reagent). B) Induced MSCs (cultured in Quantum using FN as a coating
reagent). C) Induced MSCs (cultured in Quantum using CPPT as a coating reagent). D) Induced MSCs (cultured in Quantum using VN as a coating reagent). Positive
alazarin red staining indicates the presence of calcium deposits indicative of osteogenesis.

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N.D. Frank et al.
A B
Fig. 8. Chondrogenesis (40×). A) Induced MSCs (cultured in Quantum using FN as a coating reagent). B) Induced MSCs (cultured in Quantum using CPPT as a
coating reagent). C) Induced MSCs (cultured in Quantum using VN as a coating reagent). Positive alcian blue staining indicates the presence of differentiated
chondrocytes.
5. Conclusion
This report defines 3 options for reagents that can be used to coat
the HFB of the Quantum system in preparation for culture of MSCs: FN,
CPPT, and VN. Cells harvested from Quantum using each of these 3
compounds as coating reagents met ISCT standards for plastic ad-
herence, surface marker expression, and successful trilineage differ-
entiation. No significant differences were observed among the dTs from
Quantum MSC harvests using FN, CPPT, or VN as coating reagents
(P = 0.31). Five other potential coating substances—gelatin, HSA,
collagen I, poly‑L‑lysine, and poly‑D‑lysine—resulted in poor MSC ex-
pansion and are not recommended for Quantum HFB coating.
Of the reagents that supported MSC expansion, each has a set of
associated advantages in different contexts. FN is well tested, reliable,
and available worldwide. CPPT is very effective as a coating reagent;
however, CPPT is a compound of variable composition from manu-
facturer to manufacturer, and the fact that glycoprotein content is not
typically measured during CPPT production makes it difficult to state its
composition precisely; therefore, CPPT efficacy as a coating agent may
vary between vendors. VN is widely available and has a cost and effi-
cacy that is comparable to FN in these experiments at the concentra-
tions described. The VN product used in these experiments is re-
combinant, which eliminates concern for contaminating animal
proteins. The fact that this VN product line is available in both research-
grade and GMP versions makes it an ideal candidate to facilitate the
transition from research to clinical cell manufacturing while mini-
mizing regulatory complications.
Acknowledgments
We would like to thank Rebecca Peters, Domicinda Hill, Kim
Nguyen, Brian Nankervis, and Thomas Startz for their contributions to
this manuscript.
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