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1. COLLECTION AND PROCESSING OF
HPC-C : PRACTICAL ASPECTS
DR AKSHAYA TOMAR
MD IMMUNOHEMATOLOGY AND BLOOD
TRANSFUSION
AFMC PUNE
6/18/2019

2. A QUICK OVERVIEW OF LAST LECTURE
• Hematopoietic stem cells – primitive pluripotent , used to
treat diverse array of diseases
• Main sources – BM , PBSC , CB
• Autologous vs Allogenic transplants
• Comparison between sources of stem cells
• Donor selection and collection of stem cells
• Post collection manipulation and processing of stem cells :
Volume reduction (plasma depletion); RBC depletion ;
enrichment
• Cryopreservation of stem cells – Dump freezing Vs CRF6/18/2019

3. INTRODUCTION
• Bone marrow transplantation (BMT) is an often-used means
of therapy for a variety of diseases, including chemotherapy-
resistant malignancies and genetic blood disorders .
• Lack of adequate number of HLA matched donors and high
risk of GvHD limit many allogenic transplantation
• Work that was begun in the early 1980s revealed that cord
blood was comparable to bone marrow in terms of its utility
in stem cell transplantation.
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4. INTRODUCTION
• Cord blood now a days used as
– Substitute to Bone marrow/PBSC
– A potent pluripotent stem cell source for regenerative medicine
• Advantages
– Readily available
– No risk to patient or donor
– lesser HLA restriction (4/6 match is enough)
– Lower incidence of GvHD
– No risk of infectious diseases
– Immunosuppressant requirement is minimal
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5. ESTABLISHED INDICATIONS OF UCB
• Leukemia
• Thalassemia
• Sickle Cell Disease
• Inborn Error Of Metabolism – Hurler disease , MPS
• Severe combined immune deficiency
• Experimental phase: (> 75 indications)
– Cerebral palsy ; Type I DM ; Autism ; Head Injury
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6. 6/18/2019

7. HISTORY
• 1974 – Discovered presence of progenitors in cord blood(CB)
• 1983 – Concept of using CB as alternative source of stem cells is proposed
• 1989 - First successful umbilical cord blood transplantation was reported in a
boy with Fanconi’s anemia, using umbilical cord blood of his HLA matched
sister (France)
• 1992 – First cord stem cell blood bank in New York (CryoCell International
funded by National Institute of Health) , later became the cord blood registry
in 1996
• 1993 – First private cord stem cell bank – Via Cord ,Hebron,Kentucky (USA)
• 1993- First Unrelated cord stem cell transplant
• 1995 – Cord blood registery established in San Francisco
6/18/2019

8. INDIAN CORD STEM CELL BANKS
• Life Cell – Chennai & Gurgaon
• JEEVAN – Chennai
• Stem Span – Bangalore
• CORDLIFE – KOLKATA
• REELABS – MUMBAI
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9. 6/18/2019

10. TYPES OF UCB TRANSPLANTS
UCB
RELATED
(non malignant diseases)
UNRELATED
(malignant diseases)
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11. • COLLECTION
– Signed informed consent
– Mothers tested for infectious disease(HIV,HBV,HCV etc)
– Collected samples tested for microbial sterility
– Prior to collection Umblical cord is cleaned with alcohol/betadine
• It can be of two types
– Open system (obsolete now)
– Closed System
• Closed system can be of two types
– Before delivery of placenta (done by Obstetrician In OT)
– After delivery of placenta (done by UCB collection technicians)
6/18/2019

12. COLLECTION TECHNIQUES
IN UTERO EX UTERO
•Blood is aspirated from the placental
vein while the placenta is still in utero.
•Avoids the possibility of a failed
collection
•Associated with an increase in
volume and reduced incidence of
clotted collections.
•Done by trained obstetricians
•Collections after the infant and
placenta are delivered
•Robust cleaning of the umbilical cord
and aspiration of the blood from the
placental vein.
•Handled by trained UCB bank staff in
a dedicated area outside of the
delivery room
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13. OPEN SYSTEM
CLOSED SYSTEM
(IN UTERO)
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14. COLLECTION AFTER DELIVERY OF PLACENTA
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15. MODIFIED METHOD FOR
UCB COLLECTION
•NEEDLES EXPOSURE
AVOIDED
•HIGH VOLUME AND CELL
YIELD
•LOW RISK OF
CONTAMINATION
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16. 6/18/2019

17. CORD BLOOD : VOLUME REDUCTION
• Long-term storage of unmanipulated CB donations requires
extensive and costly liquid nitrogen storage space.
• Reducing the volume of the CB unit, prior to storage, provides a
substantial reduction in
– The per unit cost of the cryogenic space
– Volumes of (DMSO) used
– Red cell content
• An added advantage of reduced volume CB product is improved
quality of product due to uniform freezing
6/18/2019

18. CORD BLOOD : VOLUME REDUCTION
• The primary objective of the volume reduction method is to reduce
all CB units to a standard volume, while maintaining the quantity,
quality, and functionality of the cells without the risk of
contamination, compatible with large-scale banking.
• Processing systems must be reproducible, reliable, and efficient.
• Reduced hematocrit in volume reduced units has been shown to
correlate with improved recovery of CFU after cryopreservation and
thawing
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19. CORD BLOOD : VOLUME REDUCTION
• Methods of volume reduction:
– Separation using density gradients
– Sedimentation
– Red cell lysis using ammonium chloride
– Centrifugation
– Filtration
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20. METHODS
HYDROXY ETHYL STARCH (HESPAN)
• The most widely used method is the hydroxyethylstarch (HES)
technique developed by Rubinstein et al. in 1995
• In order to efficiently store a large number of CBUs most of the
plasma and red blood cells are removed by differential
centrifugation
– using the ability of Hespan to induce red blood cell
agglutination.
• Recovery and concentration of the nucleated cells and progenitor
cells is achieved through two centrifugation steps
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21. EQUIPMENT REQUIRED
– Laminar Flow Hood
– Centrifuge
– Heat Sealer
– Plasma Expressor
– Bar code scanner
– Balance
– Tube stripper
– Sterile Docking Device
• Stem Cell Processing Kit (Set
#2) Pall Medical Corporation
• Hemostats
• 18 Gauge Luer Lock
• Needles Alcohol Swabs
• Sterility Testing Tubes
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22. • 1ml Syringe (2)
• 60 ml Syringe (opt)
• 30ml Syringe (2)
• Zipper-locked bag
• Sample Tubes
• CBU Collection and Receipt Form
• CBU Processing Form
• Cryovials (1-2ml)
• Six Microscope Slides
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23. • PROCEDURE
– Strip any tubing attached to the bag with blood in it back
into the bag, mix very thoroughly
– Seal off the tubing close to the bag
- Obtain 0.5 ml of product from the syringe port of the
collection bag (Bag #1) using sterile technique
- Perform automated nucleated cell count and manual
viability count
- If the volume of CBU (cord blood plus anticoagulant) is
between 65 and 85 ml, and the total nucleated cell count is
less than 6 x 108 cells, discard the CBU
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24. - Prepare three slides for a manual differential count.
- Place remainder of sample in “Unit Sample Testing Box” or
institutional equivalent for ABO and Rh testing.
• Average time taken in differential centrifugation (after
addition of HES) is about 60 min which is much less than
suspension of the product (for gravity sedimentation , 4-6 h)
• Differential centrifugation without HES
– Takes more time and number of spin
– Loss of viability , as centrifugal force used is more (uptill 3500g)
6/18/2019

25. Place the collection bag and connected stem cell processing kit in centrifuge
Adjust the centrifuge to get NC>60% or >80% MNCs
Mix the content by inverting unit 4-5 times
Sterile connect the stem cell processing kit
Calculate the volume of HES to be added (HES: CBU = 1:5 ratio )
Disinfect the sterile port and use 60ml syringe to add HES
FIRST SPIN AT 50g (as per EUROCORD protocol)6/18/2019

26. Express the supernatant again into connecting bag
Check for the recovery of MNCs/TNC in connecting bag
Temporarily close off the collection bag (second spin may be required)
Place the stem cell processing bag into centrifuge
Place collection bag on the plasma expressor and adjust processing bag on weighing
scale
Control the flow using roller clamp or hemostats, slowly express supernantant
(leukocyte rich plasma)
SECOND SPIN AT 400g (as per EUROCORD protocol)6/18/2019

27. CORD COLLECTION BAG ALONG WITH STEM CELL PROCESSING KIT USED IN DIFFERENTIAL
CENTRIFUGATION
6/18/2019

28. SEPAX SYSTEM
• Fully automated programmable cell processing device (Biosafe
Switzerland)
• First machine on the market with a dedicated application for
CB
• It consist of:
– Centrifuge
– Pneumatic tube with vaccum (to fill and empty the separation
chamber and lines)
– Collection bag including integral freezing bags
6/18/2019

29. • Uses 2 protocols:
– Use of sedimentation by HES
– Isolation of Buffy coat component enriched with HPC (no additives)
– Final volume can be adjusted in both the protocols
• UCB-HES protocol : takes 30min , final volume 20-50ml +/- 1
ml (TNC > 88% ; CD34 >96% ; high RBC and Plt depletion)
• UCB protocol : takes 20 min, final volume 10-90ml +/- 1ml
(TNC >85% ; CD34 >95%)
6/18/2019

30. FUNCTIONING OF SEPAX SYSTEM
6/18/2019

31. Advantages:
– Fully automated , closed system
– Consistent results in product volume, cell recovery
and viability
– Permit traceability due to bar code reading
– Remove the need to calculate volume by weight
of the product
– Collection of volume reduced product directly
into freezing bag
6/18/2019

32. FILTERATION
• Used in early 2000
• StemQuick™
• Stem Cell Collection Filter system (SCF)
• Procord™ - Terumo Corp,Tokyo, Japan.
• NC are trapped on the filter while the RC and PLT flow through into a drain
bag when allowed to gravitate.
• Sequential step elutes the trapped NC from the filter by flushing with an
inverted flow of a protein-rich recovery solution.
• Operation times were rapid; an average of 12 min was reported for the
SCF system
Asahi Medical Co.
Ltd., Tokyo, Japan
6/18/2019

33. • Although the filtration systems have demonstrated potential
for use in cell processing for CB banking, to date, they have
not been introduced into large-scale routine processing.
• Predominant reason for this is <80% recovery of TNC
6/18/2019

34. AutoXpress™ Platform
• Developed by Thermogenesis
• The AXP AutoXpress Platform is an automated, functionally closed, sterile
system that volume reduces cord blood to a precise 20 ml in less than 40
min, while retaining 97% mononucleated cells (MNCs).
• The system consists of a processing bag set and a metering device that
separates the three layers of CB into separate bags during the centrifugation
step.
• The metering device releases the RC discontinuously into a bag
• As the RC concentration decreases, an optosensor, measuring the light
transmittance, activates a valve that diverts a small amount of RC into the
buffy coat layer and plasma to fill the freezing bag to a preset volume
6/18/2019

35. AXPTM
Does not require HES
Fit into commonly used
refrigerated centrifuge
Processing time – 40 min
6/18/2019

36. VOLUME REDUCTION AND ENGRAFTMENT KINETICS
• No significant difference in engraftment or outcome data has been
reported after transplant with volume reduced CB units compared to
patients transplanted with non-volume reduced units.
Laughlin MJ, Eapen M, Rubinstein P, et al. Outcomes after transplantation of cord blood or bone marrow from unrelated
donors in adults with acute leukemia. N Eng J Med 2004;351:2276–2285.
Barker JN, Davies SM, DeFor T, et al. Survival after transplantation of unrelated donor umbilical cord blood is comparable
to that of human leukocyte antigen-matched unrelated donor bone marrow: Results of a matched-pair analysis.
Blood 2001;97:2957–2961.
Sanz GF, Saavedra S, Planelles D, et al. Standardized, unrelated donor cord blood transplantation in adults with
hematologic malignancies. Blood 2001;98:2332–2338.
6/18/2019

37. FICOLL-HYPAQUE SEPARATION
• Ficoll is a neutral, highly branched, high-mass, hydrophilic polysaccharide which
dissolves readily in aqueous solutions
• Ficoll is a registered trademark owned by GE Healthcare companies
• Ficoll is part of Ficoll-Paque, which is used in biology laboratories to
separate blood to its components (erythrocytes, leukocytes etc.).
• Ficoll-Paque density gradient can be adapted to very small sample volumes. Thus, it
is especially suitable for isolation of mononuclear cells from cord blood
6/18/2019

38. MATERIALS
• Anticoagulated cord blood (citrate dextrose, citrate phosphate dextrose,
citrate, heparin, or EDTA)
• PBS/EDTA: Phosphate-buffered saline (PBS) 0.5% (w/v)/2 mM disodium EDTA
• Ficoll-Paque Plus (Amersham Biosciences) or equivalent
• Bovine serum albumin/2 mM EDTA
• 500-ml Erlenmeyer flask or similar container
• 50-ml centrifuge tubes
• Centrifuge
6/18/2019

39. Transfer Anticoagulated cord blood into Erlenmeyer
flask or similar container
Dilute blood
with PBS/EDTA
in 1:2 or 1:4
ratio
Separate
mononuclear
cells
6/18/2019

40. SEPARATION OF MNCs
• Add 15 ml of Ficoll paque into 50 ml centrifuge tube
• Gently layer 30 ml dil cord blood after tilting the bottle
• Centrifuge 40 min at 400 × g, 18° to 20 ° C, without brake
• Using a Pasteur pipet, collect the mononuclear cell fraction at
the interface between plasma and Ficoll-Paque into a clean
centrifuge tube.
• Wash the mononuclear fraction with PBS
• Discard the supernatant and suspend the mononuclear cells
in 5 to 10ml of PBS/0.5% bovine serum albumin/2 mM EDTA
and proceed with cell counting.
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41. 6/18/2019

42. 6/18/2019

43. TNC AND MNC RECOVERY COMPARISON
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44. EX VIVO EXPANSION
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45. INTRODUCTION
• Limited number of (HSPCs) and prolonged time of recovery after
the transplantation are significant limitations in the use of cord
blood
• UCB contains blood cells of different commitment:
– Mature blood elements
– HSPCs
– Undifferentiated somatic stem cells
– Multipotent mesenchymal stem cells
– Endothelial progenital cells
6/18/2019

46. WHY ONLY UCB?
• In contrast to bone marrow HSPCs, UCB HSPCs are outside of
the cell cycle, but they have a pronounced and rather fast
proliferative response to growth factors stimulation – making
them ideal candidate for ex vivo expansion
6/18/2019

47. STRATEGIES
1. Expansion of committed hematopoietic progenitors (reduces
the duration of hematopoietic recovery)
2. Increasing the number of cells with a high proliferative
potential,HSPCs (eliminates the need of additional unit of
UCB)
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48. Methodological approaches for ex vivo expansion
of cord blood cells
6/18/2019

49. USE OF ENRICHED FRACTIONS OF UCB
• Separation of HSPCs is performed using magnetic or
fluorescent-labeled monoclonal antibodies against specific
antigens
• It is possible to use either a positive (isolation of certain types
of cells from heterogeneous initial material) or
negative (unwanted cells are removed from the suspension)
• CD34 and CD133 are the most common markers
6/18/2019

50. SOLUBLE COMPONENTS OF CULTURING
• Fetal Calf Serum/Fetal Bovine Serum - natural cocktail of
growth factors, adhesion mediators, minerals, lipids and
hormones, is the standard component
• Next is Cytokine cocktail - most commonly used factors are
the stem cell factor (SCF), IL-3 and -6, G-CSF, thrombopoietin
(TPO), and Flt-3 ligand
• Stem Cell Factor (SCF), IL-3 and -6, G-CSF – cell proliferation
TPO – cell differentiation and maturation
6/18/2019

51. CULTURE TECHNIQUE USING FBS AND
CYTOKINES COCKTAILS
Transfer into 20% FBS + GM-CSF,SCF,IL3,Flt3,SCF
CD34 proliferation next 7 days
Purified HSPCs
4% FBS + SCF,IL3,Flt3,TPO
First 3 days
Due to ethical and scientific issues , FBS is
not a preferred media now a days6/18/2019

52. WHEN TWO UCB UNITS ARE USED
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53. CO-CULTIVATION WITH STROMAL CELLS
• Co-culturing with stromal feeder cells is a more physiological
alternative to the application of recombinant cytokines.
• Most rational approach to the expansion of HSPC in vitro is to
use mesenchymal stromal cells as a feeder layer
• MSCs and more differentiated stromal cells secrete various
cytokines [ (SDF-1), oncostatin M, morphogenetic bone protein-4 , Flt-3
ligand, TGF-β, IL-1, -6, -7, -8, -11]
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54. PROTOCOL USED
1ST
• 7 days co-cultivation with MSC along
with hematopoietic growth factors
2ND
• 7 days of culturing in the presence of
cytokines alone
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55. CO-CULTIVATION WITH STROMAL CELLS
• This technique significantly reduces the neutrophils and
platelets recovery time
• The use of feeder layers for expansion of UCB HSPCs allows
one to exclude exogenous growth factors that reduce the
efficiency of cell amplification.
6/18/2019

56. CO-CULTURE AND SCHEMATIC DISTRIBUTION OF HSPCS
HSPCs under MSC
Adhered HSPCs
Suspended
HSPCs
Day 0
Stromal layer
CB MNCs
Day 3 Day 7
Non adhered
HSPCs
HSPC suspension
(Mesenchymal stromal cells)
6/18/2019

57. EXPANSION VS In-VIVO RECONSTITUTION
• Expanded cells are of no use if they cant engraft and
reconstitute normal hematopoiesis
• Rapid ex vivo expansion can:
– Exhaust the primitive HSC pool
– Increase the chances of genetic mutations
– Delays homing and engraftment
6/18/2019

58. The policy of the American Academy of
Pediatrics states that "private storage of cord
blood as 'biological insurance' is unwise"
unless there is a family member with a
current or potential need to undergo a stem
cell transplantation6/18/2019

59. UCB AND REGENERATIVE MEDICINE
• BASIS:
– Paracrine-based signaling facilitating endogenous tissue
repair rather than direct cell replacement or engraftment
– Immunomodulation : inflammation & immune response
6/18/2019

60. SUMMARY
• UCB is a potent alternative of HSCs (less MHC restriction)
• However, the emergence of haploidentical HSCT has resulted in a
decline in the use of UCB and a plateau in global inventories.
• Future of UCB is in regenerative medicine and adoptive
immunotherapy
• Manipulation , processing and ex-vivo expansion are important tools
of better utilization6/18/2019

61. BIBLIOGRAPHY
• Collection, Processing, and Banking of Umbilical Cord Blood Stem Cells
David T. Harris,Lab Medicine - 2008
• Factors Influencing the Umbilical Cord Blood Stem Cell Industry: An
Evolving Treatment Landscape Dessels et al,Stem cell translational medicine-2018
• Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells from
Umbilical Cord Blood Sotnezova et al, Acta Nature – 2016
• Methods of ex vivo expansion of human cord blood cells: challenges,
successes and clinical implications Baron et al, Expert review in hematology -2016
• Isolation of Mononuclear Cells from Human Cord Blood by Ficoll-Paque
Density Gradient Jaatinen et al, Current Protocols in Stem Cell Biology – 2007
• Cord Blood Processing: Volume Reduction Cell Preservation Technology -2006
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