Title: Cultivation and cryopreservation of MSCs derived from cord tissues with cord

blood AB plasma as a replacement to FBS for their potential use in therapeutics.

Author names and affiliations:

Talwadekar Manasi1,Kadekar Darshana

2,Rangole Sonal, Khan Nikhat Firdaus,Kale

Vaijayanti and Limaye Lalita

Stem Cell Laboratory , National Centre for Cell Science, NCCS complex, University of

Pune Campus, Ganeshkhind, Pune – 411007, Maharashtra, India

Corresponding author:

Dr.(Mrs.)Lalita S.Limaye,Scientist G, Stem Cell Laboratory, National Centre for Cell

Science, NCCS complex, University of Pune Campus, Ganeshkhind, Pune – 411007,

Maharashtra, India

Email- lslimaye@nccs.res.in / lslimaye@gmail.com Phone: +91-20-25708079 Fax:+91-

20-25692259

Present/permanent address:

Dr.(Mrs.)Lalita S.Limaye,Scientist G, Stem Cell Laboratory, National Centre for Cell

Science, NCCS complex, University of Pune Campus, Ganeshkhind, Pune – 411007,

Maharashtra, India

Email- lslimaye@nccs.res.in / lslimaye@gmail.com Phone: +91-20-25708079 Fax:+91-

20-25692259

Suggestions for reviewers:

1.Dr. Ramesh Bhonde, Ph.D, FNASc

School of Regenerative Medicine

Manipal University, Manipal.edu, Madhav Nagar, Manipal - 576104,Karnataka.

E-mail- rr.bhonde@manipal.edu

Official Phone:+91 80 28460671/81

2. T R Santhosh Kumar, Ph.D

Scientist E-I Cancer Research Program

Rajiv Gandhi Centre for Biotechnology

Thycaud Post, Poojappura

Thiruvananthapuram - 695 014

Kerala, India

Email- trsanthosh@rgcb.res.in

Official Phone:+91-471-2529500

3. Prof. Geeta K. Vemuganti

School of Medical Sciences,

University of Hyderabad, Central University P.O.,

Prof. C.R.Rao Road, Gachibowli, Hyderabad-500046 (Telangana), India.

Telephone : (040) 23134780

E-Mail : gkvemuganti@gmail.com / deanmd@uohyd.ernet.in

Abstract:

Objective : Neonatal tissues, Cord and Placenta are explored as alternate sources of

MSCs for their therapeutic applications. Conventionally MSCs isolated from these

sources are maintained and propagated in FBS containing medium. Serum promotes

growth and survival of cells and also protects the cells during freezing. However clinical

protocols do not encourage use of animal products. Thus, there is a need for replacement

of FBS by equally potent and clinically acceptable cost effective source.

Materials and Methods: This study is designed to compare the effect of cord blood

plasma (CBP) vs MSC qualified FBS (M FBS) during culture and cryopreservation of

MSCs. MSCs were isolated both from cord and placenta and propagated with either M

FBS or CBP. The efficiency of the cultures were analysed by growth curve, morphology,

phenotype and functionality . The cryo- protective role of the CBP was evaluated by

freezing the MSCs .

Results: The data clearly shows that CBP is equivalent in nature to M FBS for culturing

placental MSCs in terms of phenotype, proliferation rate and differentiation to different

lineages. Though Cord MSCs displayed slow growth rate, reduction in surface expression

of CD105 marker when grown in CBP all the other parameters were comparable.

Freezing of MSCs with CBP resulted in reduction in the late apoptotic cells as well as

necrotic cells.Thus CBP imparts superior protection against cryogenic insults as

compared to M FBS.

Taken together, CBP appears to be a valuable substitute to M FBS for cultivation and

freezing of MSCs.

Keywords: Mesenchymal Stem Cells, Cord, Placenta, Mesen FBS, Cord blood plasma

Abbreviations: MSC - Mesenchymal Stem Cells, MFBS - Mesen Fetal Bovine Serum,

CBP - Cord Blood Plasma, PRP - Platelet rich plasma, RPMI - Roswell Park Memorial

Institute, DMSO - Dimethyl sulphoxide, MTT - 3-(4,5-dimethylthiazol-2-yl)2,5-

diphenyltetrazolium bromide, PE – Phycoerythrin, APC – Allophycocyanin, IMDM

- Iscove’s Modified Dulbecco Medium, CFU-F - Colony forming unit fibroblast

Conflict of interest statement

All the following authors declare that there is no conflict of interest.

Authors:

1. Talwadekar Manasi

2. Kadekar Darshana

3. Rangole Sonal

4. Khan Nikhat Firdaus

5. Kale Vaijayanti

6. Limaye Lalita

Title: Cultivation and cryopreservation of MSCs derived from cord tissues with cord blood AB plasma as a replacement to FBS for their potential use in therapeutics.

Author names and affiliations:

Talwadekar Manasi1, Kadekar Darshana2, Rangole Sonal, Khan Nikhat Firdaus, Kale Vaijayanti and Limaye Lalita

Stem Cell Laboratory, National Centre for Cell Science, NCCS complex, University of

Pune Campus, Ganeshkhind, Pune – 411007, Maharashtra, India Corresponding author:

Dr.(Mrs.) Lalita S. Limaye,Scientist G, Stem Cell Laboratory, National Centre for Cell Science, NCCS complex, University of Pune Campus, Ganeshkhind, Pune – 411007,

Maharashtra, India

Present/permanent address:

Dr.(Mrs.) Lalita S.Limaye, Scientist G, Stem Cell Laboratory, National Centre for Cell

Science, NCCS complex, University of Pune Campus, Ganeshkhind, Pune – 411007, Maharashtra, India

Suggestions for reviewers:

1. Dr. Ramesh Bhonde, Ph.D, FNASc School of Regenerative Medicine Manipal University, Manipal.edu, Madhav Nagar, Manipal - 576104,Karnataka. E-mail- rr.bhonde@manipal.edu Official Phone: +91 80 28460671/81

2. T R Santhosh Kumar, Ph.D Scientist E-I Cancer Research Program

Rajiv Gandhi Centre for Biotechnology Thycaud Post, Poojappura Thiruvananthapuram - 695 014 Kerala, India Email- trsanthosh@rgcb.res.in Official Phone:+91-471-2529500

3. Prof. Geeta K. Vemuganti School of Medical Sciences, University of Hyderabad, Central University P.O., Prof. C.R.Rao Road, Gachibowli, Hyderabad-500046 (Telangana), India. Telephone : (040) 23134780 E-Mail : gkvemuganti@gmail.com / deanmd@uohyd.ernet.in

ABSTRACT

Objective: Neonatal tissues, Cord and Placenta are explored as alternate sources of

MSCs for their therapeutic applications. Conventionally MSCs isolated from these

sources are maintained and propagated in FBS containing medium. Serum promotes

growth and survival of cells and also protects the cells during freezing. However, clinical

protocols do not encourage use of animal products. Thus, there is a need for replacement

of FBS by equally potent and clinically acceptable cost effective source.

Materials and Methods: This study is designed to compare the effect of cord blood

plasma (CBP) vs MSC qualified FBS (M FBS) during culture and cryopreservation of

MSCs. MSCs were isolated both from cord and placenta and propagated with either M

FBS or CBP. The efficiency of the cultures was analyzed by growth curve, morphology,

phenotype and functionality. The cryo-protective role of the CBP was evaluated by using

it in freezing medium of MSCs.

Results: The data clearly shows that CBP is equivalent in nature to M FBS for culturing

placental MSCs in terms of phenotype, proliferation rate and differentiation to different

lineages. Though Cord MSCs displayed slow growth rate, reduction in surface expression

of CD105 marker when grown in CBP all the other parameters were comparable.

Freezing of MSCs with CBP resulted in reduction of the late apoptotic as well as necrotic

population. Thus CBP imparts superior protection against cryogenic insults as compared

to M FBS.

Taken together, CBP appears to be a valuable substitute to M FBS for cultivation and

freezing of MSCs.

Keywords: Mesenchymal Stem Cells, Cord, Placenta, Mesen FBS, Cord blood plasma

Abbreviations: MSC - Mesenchymal Stem Cells, MFBS - Mesen Fetal Bovine Serum,

CBP - Cord Blood Plasma, PRP - Platelet rich plasma, RPMI - Roswell Park Memorial

Institute, DMSO - Dimethyl sulphoxide, MTT - 3-(4,5-dimethylthiazol-2-yl)2,5-

diphenyltetrazolium bromide, PE – Phycoerythrin, APC – Allophycocyanin, IMDM

- Iscove’s Modified Dulbecco Medium, CFU-F - Colony forming unit fibroblast

INTRODUCTION

Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells with multi-

differentiation capacity. MSCs have gained a substantial therapeutic value because of

their plasticity and immune suppressive characteristics (1). MSCs are known to be

isolated majorly from bone marrow aspirates. Owing to the invasive procedure and

limitation in amount of sample that can be procured, alternate sources such as umbilical

cord, placenta, endometrial polyps, menses blood, adipose tissue, etc. are being harnessed

to obtain MSCs (2).

Cord / Placenta derived MSCs need to be cryopreserved as and when available so that

they can be revived and used in clinics whenever required. Generally most protocols

describe use of specialized FBS like mesenFBS (M FBS) for culturing and freezing

MSCs (3, 4). However, clinical transplantation protocols demand replacement of

xenogeneic products as these may cause adverse immunological reactions and may also

transmit infectious agents (5). Many investigators have used serum free media for culture

and expansion of MSCs (6). But these are expensive and not as potent as serum

containing media. Thus there is a need for substitutes for xenogeneic sera like autologous

serum (7), plasma (8), PRP (9), platelet lysates (10). We here describe use of cord blood

derived AB positive plasma (CBP) for both culture and freezing of MSCs isolated from

cord (C MSCs) and placenta (P MSCs) and compared its efficiency with mesen FBS (M

FBS). The data prompts us to state that it can serve as a valuable substitute to FBS and

will thus become more acceptable in the clinics.

MATERIALS AND METHODS

Isolation of mesenchymal stem cells (MSCs)

Umbilical cord and placenta were collected from full term deliveries from clinics. The

collection was performed with informed consent and as per the protocols approved by the

Institutional Ethical Committee -NCCS, Pune. Umbilical Cord and a section from the

central part of the placenta were used to isolate the Mesenchymal stem cells. The tissues

were washed with plain IMDM (Sigma Aldrich, St Loius) and chopped into pieces and

then subjected to enzymatic digestion with 0.25% Trypsin for 30 minutes. Single cell

suspension was obtained by passing the homogenate through sterile muslin cloth. The

cells were washed with medium and suspended in complete growth medium (RPMI 1640

(Sigma Aldrich, St Loius) with 20 % M FBS (Gibco, Invitrogen) and were seeded in 75

cm2 tissue culture flasks (Falcon, Beckton Dickinson, San Jose, CA). Non-adherent cells

were removed after 72 hours. Cultures were re fed with fresh medium after every 72 hrs

for 10-20 days. The cultures were maintained by passaging them after reaching 70-80%

confluency, for 6-7 passages. The cells between passage numbers 3-6 were used for the

experiments after confirming the phenotypic signature of the MSCs.

Collection of AB positive plasma from cord blood

Cord blood samples were collected from local hospitals with the compliance of the

institutional review board (IEC-Institutional ethical committee, NCCS). Blood grouping

was carried out with ERYSCREEN reagent (Tulip diagnostic, India). From the AB

positive samples, mononuclear cells were isolated by ficoll hypaque density gradient

method. (Density 1.077 g/ml, Sigma Aldrich, St. Louis MO). Separated plasma was

collected and inactivated at 560C for 30 mins. Frozen aliquots were stored at -200C and

further used for maintenance of MSCs.

Cultivation of C and P MSCs with M FBS and CBP

MSCs (n=3) in passage 3 were harvested and re cultured in two sets, one with

RPMI+20% M FBS and second set with RPMI+20%CBP, with equal seeding density in

T25 cm2 flasks. After the cells reached confluency they were harvested and used for

subsequent experiments.

Growth kinetics study

103 cells were seeded in 96 well plates in medium with M FBS/CBP. Proliferation was

assessed by MTT assay. The cultures were followed till 6 days i.e.144 hrs. Absorbance

was measured at 570nm.

Characterization of MSCs by flow cytometry

MSCs expanded from cord / placental tissue were characterized by using following

antibodies CD105-PE, CD73-APC, CD166-PE, CD90-APC, CD34-PE (BD Pharmingen-

San Jose, California) and CD45-APC (eBiosciences, San Diego USA). Isotype matched

antibodies were kept as controls. The fluorescently labeled cells were acquired on FACS

Canto II Aria (BD). An acquisition of 10000 events was done and data was analyzed by

FACS DIVA - version 5.0

Colony Forming Unit -Fibroblast assay (CFU-F)

5 x 103 MSCs were seeded in 60 mm dish with M FBS/CBP containing medium and

incubated at 37°C, 5% CO2 humidified environment for 7-10 days. After that, non-

adherent cells were removed, the monolayer was washed with PBS and fixed using 100%

methanol (Fischer Scientific) for 10 min. Staining was done with 0.1% crystal violet

solution for 10 mins. Clones of >50 cells were scored as CFU-F. Staining was done in

triplicates for each sample.

Differentiation to osteoblasts and adipocytes.

104 MSCs were seeded in to 24 well plates (Falcon, Beckton Dickinson, San Jose, CA)

and grown till 60-70% confluency. The cells were then subjected to osteogenic and

adipogenic differentiation for 15- 18 days using kit (STEMPRO®

osteogenesis/adipogenesis Differentiation Kit, Invitrogen). The osteogenic and

adipogenic differentiation was confirmed by performing Alizarin Red S (Sigma Aldrich)

staining for calcium deposits and intracellular lipid droplets staining with Oil red O dye

(Sigma Aldrich) respectively.

Cryopreservation of MSCs

106 MSCs were cryopreserved in conventional freezing medium containing 10% DMSO

+ 20% MFBS/Cord blood plasma by portable programmable freezer (Freeze Control,

Australia) at controlled cooling rate of 10C /min down to -40oC followed by 100C/min

down to -90oC and then located in liquid nitrogen. The cells were thawed by rapidly

immersing the vials in a water bath at 370C. The viability of the cells was assessed by

trypan blue dye exclusion method. Further, apoptotic profiling was carried out by

performing Annexin V and PI staining (BD Biosciences) as per manufacture’s instruction

and analyzed by flow cytometry.

Statistical analysis

The statistical differences between groups (n=3) were analyzed by one way repeated

measure analysis of variance using the software SIGMA STAT (Jandel Scientific

Corporation, San Rafael, CA) for all the experiments. The values were plotted as mean

±standard deviation Probability value: *p≤0.05, **p≤0.01, & ***p≤0.001 were

considered statistically significant.

RESULTS

Morphological differences in MSCs cultured in M FBS vs CBP

MSCs could be cultured in both M FBS and CBP and the cells showed typical

fibroblastic morphology. However, with both C MSCs and P MSCs, the growth in CBP

was aberrant and persistent clusters were seen in the cultures. These clusters were more

prominent in C MSCs (Fig. 1 A) as compared to P MSCs.(Fig. 1 B) indicating a delayed

growth in the former as compared to the latter. This pattern of growth in C MSCs may be

attributed to low seeding density as this trend was reversed by increasing seeding

densities.

C-MSCs grown in FBS show higher proliferation rate

In order to check whether the differences in morphological appearances of the cultures

were reflected in their proliferation rates we studied growth kinetics of the MSCs in M

FBS vs CBP. As expected C MSCs with M FBS showed higher growth rates as

compared to those grown with CBP and the difference was significant at day six(Fig

1C).However P MSCs showed more or less similar proliferation patterns with

FBS/Plasma (Fig. 1 D).

P MSCs grown in M FBS/CBP show equivalent level of expression of surface markers

We next proceeded to characterize the MSCs grown with FBS/Plasma for the expression

of an array of surface markers. No significant differences were observed with P MSCs /C

MSCs in both media for different surface marker expression like CD73, CD90 and CD44.

However, C MSCs showed 50% less expression of CD105 when grown in AB plasma.

Reduction in CD105 expression is also reported by Mark et al in serum free media using

human bone marrow samples (11).Histogram overlays for a panel of antibodies are

depicted in Fig. 1 E and F. for C and P MSCs respectively. The MFI levels were

strikingly higher in C MSCs with M FBS as compared to those seen in CBP whereas in P

MSCs this difference was marginal.

MSCs cultured in cord plasma have comparable CFU-F potential and differentiation

ability

The clonogenicity of MSCs was assessed by CFU-F assay. MSCs in cord plasma showed

120±5 CFU-F and in M FBS there were 150±10 per 5x103 cells seeded. Thus, the CFU-F

potential was comparable in both the media. Differentiation of MSCs to adipocytes and

osteoblasts was similar in both M FBS and CBP containing media. Images of CFU-F

(Fig.2A) and differentiation to adipocytes (Fig.2B) and osteoblasts (Fig.2C) of

representative MSCs in M FBS and CBP are depicted.

Cord plasma is a useful substitute to FBS for cryopreservation of MSCs

The cells are subjected to stress during cryopreservation and this stress may be enhanced

if the culture medium and freezing media are different. So we decided to freeze them in

the same media. C and P MSCs grown with M FBS and CBP were cryopreserved in

respective media containing 10%DMSO and 20% FBS/ Plasma .The viability post revival

as checked by Trypan blue dye exclusion level ranged from 75 to 80 % in all the sets.

Cryopreserved MSCs were revived and assessed for retention of cell surface markers .No

significant differences were observed in the two sets except for the expression of CD105

in C MSCs (Fig 3A).

The revived MSCs from the two sources frozen with two different media were subjected

to annexin V –P.I staining and analyzed on flow cytometry for the percentage of viable,

apoptotic and necrotic cells. C MSCs frozen with FBS showed less viable cells as

compared to M FBS whereas P MSCs showed similar number of viable cells in the two

sets. Lesser number of early apoptotic cells and more late and necrotic cells were

observed in both P and C MSCs frozen with M FBS as opposed to those frozen with CBP

(Fig 3B). A representative FACS profile is depicted in Fig 3C. This data suggests that

CBP is more efficient than M FBS in protecting the MSCs from cryo injuries.

DISCUSSION

Bone marrow MSCs are difficult to obtain and their quality deteriorates with age. So

cord tissue derived MSCs are becoming popular these days for cell based therapies (2).

MSCs are generally propagated in serum containing media. Because of the xenogeneic

nature of FBS, there is an elevated risk of transmitting infectious agents and adverse

immunological reactions (3, 4). In the current work we attempted to culture and

cryopreserve clinically compliant MSCs by replacing M FBS with AB positive cord

blood plasma in the media. Cord blood is a clinical waste material and easy to procure

thus CBP is a cost effective substitute for FBS. We report the use of CBP for cultivation

and cryopreservation of MSCs isolated from both cord and placenta. We preferred

plasma over serum to curtail the loss of precious stem cells with clotted fraction while

collecting serum. AB positive plasma was selected as it is a universal recipient blood

group.

Our data convincingly shows that CBP is as effective as M FBS with respect to important

characteristics like growth pattern, phenotypic signature, clonogenic ability and

differentiation capabilities of both C MSCs and P MSCs. However the observed lag in

the growth kinetics for C MSCs in FBS can be attributed to the delay in the adaptation to

the serum replacement. The lag period was shortened by increasing the seeding densities

during the initial passages. On the contrary, P MSCs showed comparable growth in both

the media. The immunophenotype of the both MSCs was also found to be similar except

for reducedCD105 expression by C MSCs in CBP. Our data is in agreement to those of

Mark et al (11) who have reported decrease in the CD105 expression in the serum free

medium as compared to serum containing media. The reason for PMSCs not showing this

reduction in expression of CD105 may be attributed to their more adaptable nature.

Cryopreservation of MSCs in chemically defined media is essential for their application

as ready to use, off the shelf cell products in therapeutics. Roy et al (12) have tried to

replace serum by sucrose during cryopreservation of MSCS and report that the cells are

slightly compromised than the fresh cells. We here report the use of CBP as a superior

cryo- protectant over conventional FBS in the freezing medium. Substitution of FBS with

CB plasma in the freezing medium in our studies had no adverse effect on viability of

the cells as detected by both Trypan Blue dye exclusion and annexin PI staining.

Interestingly, MSCs frozen with CBP exhibited enhanced protection from cryo-injury, as

there was a significant reduction in the late apoptotic and necrotic population as opposed

to MSCs frozen with FBS. Though we observed an increase in the early apoptotic

population for CBP set as compared to FBS set, this population can be rescued back to

the Annexin V- PI- (viable) phenotype by continual maintenance at 370C (13,14).

Recently Ping et al (4) in biomaterials describe the use of fetal ECM proteins for

culturing adult MSCs thus giving them natural biological supports as compared to

synthetic polymers With an increasing trend towards the use of natural non xenogenic

substitutes for culture, cryopreservation and expansion of MSCs, the use of cord blood

plasma stands as a potential, economical and valuable substitute for FBS in culture and

freezing media for MSCs. However now it remains to be seen whether adaptation of the

cultures for a few passages will further improve their growth patterns and expression of

surface molecules while retaining their genetic stability.

Taken together, our data points to AB+ cord blood plasma as a clinically relevant

substitute for FBS.

ACKNOWLEDGEMENT

We thank BRNS, DAE, Mumbai for funding the project. Authors 1,2 receive fellowship

from UGC, New Delhi. We thank local hospitals like Deenanath Mangeshkar Hospital,

Pune for providing Cord blood samples, Dr. Prakash Daithankar from Onkar Hospital,

Pune for providing clinical samples like cord and placenta. We thank FACS facility of

NCCS for acquiring samples on Flow cytometer.

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Fig 1. Morphology, growth kinetics and phenotype of MSCs grown with M FBS &

CBP Phase contrast images of C MSCs and P MSCs grown in M FBS (A & C) and CBP

(B & D) respectively. Comparison of growth kinetics of C MSCs (E) and PMSCs (F) in

M FBS and CBP. Surface marker analysis by flow cytometry of MSCs grown in either M

FBS or CBP in both CMSCs (G) and PMSCs (H). ***p≤0.001

Fig 2. In vitro functionality of MSCs is unaltered by CBP

(A) Distribution of colonies of MSCs in CFU-F cultures grown in MFBS and CBP.

MSCs cultured in M FBS and CBP were subjected to (B) adipogenic and (C) osteogenic

differentiation and stained with oil red O and Alizarin red S respectively.

Fig 3. Freezing of MSCs with M FCS/CBP (A) Post thaw phenotypic analysis revealed

no change in expression of positive markers like CD44, CD73, CD90 except CD105 (B)

Annexin V staining shows significant decrease in the late apoptotic and increase in the

early apoptotic population when MSCs were frozen with CBP (C) Representative FACS

profiles of Annexin V and PI staining for MSCs frozen /revived with MFBS and CBP

respectively . *p≤0.05, **p≤0.01