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A Novel Strategy for the Expansion of Peripheral Blood Stem Cells in Autologous Patient Serum, including a Proposed Design for Automation Ex Vivo
PAUL FADUOLA
A THESIS SUBMITTED IN PARTIAL FULFILMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
DO C T OR O F PHI L OSOPH Y
IN
STEM CELL BIOLOGY
PANAMA COLLEGE OF CELL SCIENCE
2014
DEDICATION
To my beautiful family, i love you all.
A Novel Strategy for the Expansion of Peripheral Blood Stem Cells in Autologous Patient Serum, including a Proposed Design for Automation Ex Vivo
Paul Faduola
Stem Cell Biology
Panama College of Cell Science
Abstract
Stem Cells are now the favorite cells used for cell therapy in many disease conditions
including spinal cord injury, stroke, Alzheimer’s disease, Parkinson’s disease, and
several other problems. They are also a standard of care in the treatment of some
selected patients with cancer after high-dose chemotherapy (HDC). This is because of
their potential to repair and regenerate damaged cells, tissues and organ. The bottle
neck to the clinical application of Peripheral Blood Stem Cells (PBSCs) has been the
low level of the concentration recovered after apheresis that is mostly insufficient to
produce therapeutic efficacy. Ex vivo expansion of PBSC is a logical step to overcome
such problem, but current efforts in this direction are still inefficient.
The purpose of this thesis is to demonstrate the efficacy of scaling up of PBSC in
manipulated autologous serum coupled with the synergistic action of hematopoietic
growth factors (HGFs) in the presence of stromal cell derived factor.
We derived PDGF from autologous serum using cellaid protocol, then supplement the
serum with interleukin 3 (IL-3), interleukin 6 (IL-6), stem cell factor (SCF), granulocytes
colony stimulating factor (G-CSF), megakaryocytes growth and differentiation factor
(MGDF) and stromal cell derived factor (SCDF)
Our result indicates that bench scale expansion of PBSC is possible in autologous
serum. To translate this success into the clinic, the cells need to be expanded in a large
scale. Here we describe a proposed design for automation ex vivo that will allow our
expansion protocol to be translated into machine/device manipulation to allow for
routine clinical application that favors good manufacturing practice
Keywords:Peripheral,Blood,StemCell,Autologous,Serum,Expansion,Ex
vivo,Hematopoietic,Pluripotent,passage,Cytokines,Bioreactor,Transplant.
TABLE OF CONTENT
Abstract ............................................................................................................................3
Table of Contents ............................................................................................................5
Introduction.......................................................................................................................6
Aim of study......................................................................................................................9
Materials and Methods...................................................................................................10
Results............................................................................................................................16
Discussion......................................................................................................................17
Bioreactor System..........................................................................................................17
Moving in-vitro expanded autologous PBSC into the clinic............................................18
Novel elementary strategy for routine clinical application...............................................20
Conclusion......................................................................................................................23
Acknowledgements ........................................................................................................24
Reference.......................................................................................................................25
Tables ............................................................................................................................30
Figures ...........................................................................................................................36
Abbreviation....................................................................................................................47
Introduction Significant progress has been made lately to position stem cell therapy in clinics. Some
stem cell therapies have already been approved by regulatory bodies like FDA while
many others are either in early or late stage of clinical trials. This is not surprising
considering the potential of stem cells to repair and regenerate damaged tissues and
organ (1). Amongst the different sources of stem cells, peripheral blood stem cells
(PBSCs) are now the most commonly used for transplantation in patients above
20years (Fig 1). The advantage of PBSC as the only stem that constantly travel from
the bone marrow to areas that are damaged in the body through the peripheral blood
circulation is creating opportunities for it applications toward systemic diseases. It is
expected that the expansion and differentiation of PBSC into Mesenchymal stem cells,
muscle stem cells or neural stem cells, will provide treatment for incurable conditions
like skeletal dysplasia, Duchene muscular dystrophy and neurodegenerative diseases
like amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease (AD).
When PBSCs were discovered in 1950s, initial thought was that they are non-leukemic
DNA-synthesizing cells capable of self-renewal. These cells were later revealed to be
stem cells that originated from the bone marrow (BM) through a test called shielding
experiment. This experiment demonstrates that after total body irradiation, stem cells
from shielded areas that are rich in hematopoietic stem cells (HSC) re-entered the blood
stream and even replenish the BM. The self-renewal potentials of PBSC became the
point of attraction to scientists. Other experiments like post radiation parabiosis and
cross circulation experiments in large animals were performed to verify this regenerative
potential. They soon came to the conclusion that PBSCs were indeed capable of
replenishing the BM after chemotherapy or radiotherapy. This discovery means that
white blood cells (WBCs) could be recovered from whole blood with the possibilities that
PBSCs will be present in them. Because of the ease of collection, peripheral Blood
Stem Cell Transplantation (PBSCT) was expected to be widely acceptable and
applicable. This expectation did not materialize early due to low concentration of the
stem cells in the blood stream. A breakthrough came in timely with the invention of a
continuous-flow apheresis technology called NCI-IBM blood cell separator in the
1960s.The machine made it possible to process more of the patient’s blood to allow
more of these stem cells to be separated. This was thought to be a crucial step in
getting PBSC into the clinic, but it was not to be because the stem cell recovery from
peripheral blood was still less than the recovery from BM. Though attempts at
transplanting these cells were done, initial outcomes from transplants failed with no
recovery of granulocytes or platelets two months post transplantation. Concerns were
immediately raised about the self renewal properties and proliferative ability of these
cells. This engraftment problem was soon linked to the dose of stem cells being infused.
Another attempt at improving the amount of PBSC available for transplant was achieved
using liquid nitrogen to store these cells until a sufficient dose is harvested. Immediately
after this possibility came to light, successful transplants were achieved with fast and
complete replenishment of the hematopoietic system (2- 4).
Even though the collection of PBSC was less invasive, collecting therapeutic dose
required several apheresis to be performed making the procedure laborious. To address
this challenge, ways to improve the concentration of the stem cells temporally before
apheresis were considered. One of the ways adopted was the use of hematopoietic
growth factors to increase the concentration in the peripheral blood. This was made
possible by discovery that granulocyte colony stimulating factor (G-CSF) and
granulocyte-macrophage colony-stimulating factor (GM-CSF) initially used to manage
the side effects resulting from the reduction of blood cells due to the myeloablative
treatment can also mobilize significant amount of CD34+ hematopoietic stem cell from
the BM to the peripheral blood (5). Current efforts have aimed at ex vivo expansion to
shorten the time for effective repopulation in vivo. This includes the use of cytokines (6-
13), stromal feeder layers or extracellular matrix culture (14-16), regulatory pathway
manipulation (17-21) and chemical compound supplementation (19). None of these
approaches has effectively allowed for clinical scale ex vivo expansion of PBSC.
The time has come for PBSC ex vivo expansion to be translated into bioreactor system
for automated processing in community health centers. This step is necessary to mimic
in vivo environment effectively so that cancer patients and patients needing stem cell
therapy can have sufficient cells for transfusion (22). In cancer patients for example, not
all of them have benefitted from the conventional form of PBSCT. This is because
patients respond differently to mobilizing agent in term of PBSC expansion in the blood
stream. While some patients require a maximum of two collections to achieve
therapeutic effect, other respond poorly even after several collections. This poor
response have been linked to advanced disease status with marrow involvement at the
time of mobilization, extensive prior treatment with chemotherapy and/or radiotherapy,
prolonged disease history, low-grade histology and use of fludarabine-containing
regimens prior to mobilization (22-24). In this poor responder group, ex vivo expansion
is necessary to achieve therapeutic effect (25).
In term of stem cell therapy, PBSC harvested from the peripheral blood have shown
promise in the treatment of many systemic diseases. Stem cells have been expanded
and differentiated into other cell types and used to replace tissue that is damaged by
disease or injury. This form of therapy has been used to replace cells damaged by
spinal cord injury (26-28), stroke (29-30), heart damage (31-35), Parkinson’s disease
(36-37) and several other problems. Unfortunately, only little has been achieved in term
of technologies that will enable efficient and consistent ex vivo expansion of stem cells
to scale up its application in clinics.
AIM OF THE STUDY
The purpose of this thesis is to demonstrate the efficacy of scaling up PBSC in
manipulated autologous serum coupled with the synergistic action of hematopoietic
growth factors (HGFs) in the presence of stromal cell derived factor. We derived PDGF
from autologous serum using cellaid protocol, then supplement the serum with
interleukin 3 (IL-3), interleukin 6 (IL-6), stem cell factor (SCF), granulocytes colony
stimulating factor (G-CSF), megakaryocytes growth and differentiation factor (MGDF)
and stromal cell derived factor (SCDF). We included stromal cell derived factor as part
of the supplementation based on the evidence that extracellular matrix proteins
encourage self-renewal and adhesion of HSCs to progenitor cells. Our choice of
manipulated autologous serum is an effort to move PBSC from bench to clinic. Another
purpose of this study is to describe how this expansion protocol can be translated into
machine/device manipulation to allow for routine clinical application that favors good
manufacturing practice.
MATERIALS AND METHODS
Materials
Consumables
FercomApS - Virumvej 90 A - 2830 Virum - Denmark
Falcon Tubes 15 ml & 50 ml Falcon
Forceps
Glass Pasteur Pipettes
Glass slides
Pipette Tips 10µl, 100µl, 500µl,
Plastic Pasteur Pipettes
Scalpels Feather
Syringes, 1 ml, 2 ml, 5 ml Braun
Sterile filters 0.22µm millipore
Surgical Tweezers
Tissue Culture & Multiwell Plates BD Falcon
35-mm Petri dish Nunc
C.C. Obi Nig. Ltd, 161, Herbert Macaulay Street, Yaba, Lagos, Lagos Surgical cap
Surgical mask
Gloves
Equipment
FercomApS - Virumvej 90 A - 2830 Virum - Denmark
Autoclave
DM IRB Leica inverted microscope
Fridge & Freezer Combination
Hot Cabinet Heraeus
Incubator Thermo Scientific
Laboratory Centrifuge Heraeus
Melting-press machine
K-SYSTEMS .KivexBiotecLtd .KlintehøjVaenge1. DK-3460 Birkerød . Denmark
G 185 tri-gas incubator
IVF L426Dual Lamina Flow Cabinet
C.C. Obi Nig. Ltd, 161, Herbert Macaulay Street, Yaba, Lagos, Lagos Waterbath
Hemocytometer
Kits
Amersham Biosciences AB Björkgatan 30, SE-751 84 Uppsala, Sweden
Ficoll-paque
StemCell Technologies. United Kingdom
EasySep® Human CD34 Positive Selection Cocktail
EasySep® Magnetic Nanoparticles
MethoCult SF
JMS Co., Ltd ,Hiroshima, Japan
Cellaid® human serum collection kit
Cell culture media, supplements and reagents
Zayo-Sigma Chemicals Ltd. Jos, Nigeria
Recombinant human interleukin 3
Recombinant human interleukin 6
Recombinant human stem cell factor
Recombinant human megakaryocytes growth and differentiation factor
Recombinant human granulocytes colony stimulating factor
Recombinant Stromal cell derived factor
HTDS International, 3 rue du Saule Trapu – BP 246 91882 Massy Cedex France
Dulbecco’s Phosphate buffered Saline
C.C. Obi Nig. Ltd, 161, Herbert Macaulay Street, Yaba, Lagos, Lagos Wright stain
Giemsa stain
�
Methods
Mononuclear Cell preparation
Ethical permission was received before the donor was recruited.10mls of venous blood
was collected from the after mobilization with G-SCF. The concentration of the white
blood cell was determined manually using a hemocytometer and adjusted to 1-2 x
106cell/ml with PBS before 4mL of the cells were layered on 3mL of the Ficoll-Paque
and centrifuged at 400g for 25 minutes at 24°C. The Peripheral Blood Mononuclear Cell
(PBMNC) interface band were harvested, wash twice with phosphate-buffered saline
(PBS). This was followed by CD34+ cells selection using human CD34 selection kit.
Manual EasySep® CD34+ Cell Positive Selection using Purple EasySep® Magnet
They concentration of the PBMNC was adjusted to 2 x108 cells/ml with PBS and placed
in 5mL falcon polystyrene round tubes. 100µl of the EasySep® positive selection
Cocktail was added to 1mL of the cells. This was mixed well and incubated at room
temperature for 15 minutes.100µl of EasySep® magnetic nanoparticles was then added
to 1mL of cells, mixed well and incubated at room temperature for 10 minutes. The cell
suspension was then adjusted to 2.5mL with PBS, mixed thrice in the tube by pipetting
up and down and placed in a magnet (Fig 2) with the cap of the tube off. After 5
minutes, the magnet and the tube were inverted in one motion for 3 seconds to discard
the supernatant and then returned to the upright position leaving the magnetically
labeled cells inside the tube. The tube was then removed from the magnet and the cells
were diluted with 2.5mL of PBS with gentle pipetting up and down. The rinsing of the
magnetically CD34+ labeled cells was continued as detailed in the protocol. The CD34+
positively selected cells were then re-suspended in 2mL of PBS.
Culture Medium Preparation
The culture medium was prepared under a lamina air flow hood (Fig 8) with the same
donor’s serum and separated based on the cellaid protocol. 200mls of venous whole
blood was collected in a bag containing glass beads and incubated at room temperature
for 30minutes with constant gentle agitation to allow for platelet attachment. The serum
was separated from the whole blood by centrifuging at 2500rpm for 10 minutes and heat
inactivated at 560c for 30 minutes. This serum was then used to dilute IL-3 (10ng/mL),
IL-6 (10ng/mL), SCF (100ng/mL), G-CSF (100ng/mL), MGDF (100ng/mL) and SCDF
(100ng/mL).
Bench Scale Ex Vivo Expansion Protocol
The CD34+ positively selected cells were expanded ex vivo in a freshly prepared culture
media described above. It is generally believed that when cells are seeded in lower
concentration, they tend to expand more. We initiated our seeding at a concentration of
5000/mL of the culture media in 16 well culture plates in triplicates. The culture plates
were placed inside a G-185 tri gas incubator (Fig 9) at a temperature of 37oC, 5% CO2
and 5% O2 for 14days.
Assessment of Cell Culture
The culture assessment was done on day 7, 10 and 14 to determine the % increase,
CFU-GM numbers and morphology change of the seeded cells. Cells from at least 2
wells were washed once in PBS and used for the analysis on selected days.
Percentage Increase Check
The % increase was determined after filling the hemocytometer chamber with a pipette
containing the washed cells. A cover slip was properly mounted on the hemocytometer
to allow the fluid containing the cells to enter the chamber by capillary action when the
pipette came in contact with the edge of the cover slip. Care was taken to ensure air
bubbles were not formed and the fluid did not overflow into the trenches. The cells were
counted as described in the hemocytometer manual. The % increase was obtained by
comparing the concentration of cells seeded before and after culture.
CFU-GM Assay
This assay was used for the measurement of clonogenicity in the PBSC culture. A
35mm petri dish containing MethoCult SF was used. 1x 103 expanded cells (day 7)
,1x104 expanded cells (day 10) and 1x106 expanded cells (day 14) were seeded on
cytokine supplemented with methylcellulose and cultured in a G 185 tri-gas incubator at
37°C ,5% CO2, 5% O2 for 14 days. The culture was done alongside 700 freshly isolated
CD34+ cells/plate in methylcellulose for 14days as control. The numbers of CFU-GM
were counted in an inverted light microscope (Fig 10).
Morphology Assessment
Morphological evaluation was done on 200 differential cell counts to determine
morphological changes. This was achieved by staining slides prepared from the cells
with Wright-Giemsa stain. The stained slides were assessed for myeloid precursor cells
based on the characteristics summarized (table 1).
RESULT
Percentage increase on seeded cell in culture
The percentage CD34+ recovery in experiment 1 was 32.3%, experiment 2 was 29.5%
and experiment 3 was 35.0. There was a mean percentage increase of 66.7%, 128.7%
and 206% on day 7, 12 and 14 respectively (Table 2).
Rate of CFU-GM Expansion
They mean percentage increase in CFU-GM peak on day 7 by 69.5% above the initial
CFU-GM. We observed a 47.5% and 32.6% decline on day 12 and 14 respectively
(Table 3).
Morphological changes in cultured myeloid cells
Apart from myeloblasts that decreased on day 7 and 12, there was a mean increase in
promyelocytes, myelocytes/metamyelocytes on day 7 and 12 (Table 4).
DISCUSSION
Bioreactor System
Tissue culture flask and well plates have been the mainstay of stem cell culture and
expansion. Their wide acceptance globally was mainly due to the fact that they are
cheap and easy to use. Despite these advantages, automated bio-processing platforms
are better positioned to encourage culture and expansion of stem cells for clinical
applications. This is because they can guarantee efficient, robust and large scale
production.
Bioreactor designs are being explore to allow stem cell expansion in clinical scale. The
efficiency of such a design will depend on its ability to control how cytokines and other
growth factors are fed into the system and how the products of their metabolism are
eliminated. An efficient bioreactor system is expected to mimic in vivo scenario. To
achieve this, the system must have a very good control of aeration, temperature, light
and PH compatible to physiological life. Expansion capacity and quality can be
improved upon by understanding the appropriate mode of introducing the nutrients,
proper concentration and composition of the growth factors to be used for the
expansion. The application of such knowledge in bioreactor system will be a step in the
right direction. Presently, nutrients are introduced into the system in batch, fed batch or
continuous and perfusion. Perfusion strategy is more widely applied because it ensures
continuous renewal of nutrient and removal of metabolites from the system which is a
solid requirement for self-renewal and differentiation of stem cells. The fed-batch
method however has the advantage of supplying the nutrient in a more appropriate
manner than the perfusion strategy thus ensuring that their release and utilization are
better controlled. In this way, less metabolite that is inhibitory to expansion will be found
in the culture system.
Several bioreactor designs like stirred culture vessels (Fig 3), cell culture bags (Fig 4),
bubble column or air-lift vessels (Fig 5), rotary cell culture systems (Fig 6) and
microfluidic devices (Fig 7) have been developed. The bioreactor design that will
improve the expansion of PBSC should have an efficient control of the
microenvironment. Apart from the micro environment, more efforts should be made to
know the growth factors that improve expansion synergistically with the different
designs. Each design has its advantage and disadvantage (table 5), choosing the
appropriate culture design is therefore very critical.
Moving Ex Vivo Expanded Autologous PBSC into the Clinic
Apart from the capacity of stem cells to repopulate, they are also expected to be safe for
patient use. To fulfill this requirement, the expansion must be free of feeder cell, animal
proteins or microbial agents that might contaminate these cells. The expansion of stem
cells have since been done using different cytokine combination and concentration,
autologous serum, serum free media, fetal bovine serum, different mode of culture and
varied initial culture density. The heterogeneous nature of these trials have made
conclusion very difficult considering that one variable could have significant effect on the
outcome. Traditionally, stem cells have been cultured in FBS because they are rich in
attachment, growth factors, nutritional and physiochemical compounds that support cell
growth. However, translating this process to clinics is being hampered by the genuine
fear of contamination with undesirable pathogens such as viruses, mycoplasma, prions,
and other zoonotic agents. The use of serum free media has been suggested as a
possible way to overcome the danger of FBS. They problem with serum free media is
that they may only support expansion for single passage. Even when they are able to
support expansion for multiple passages, the rate of growth is slower. Human serum
has been offered as suitable alternative to the safety ridden FBS and the poor efficiency
serum free media. Types of human serum that have been studied include; autologous,
allogeneic and cord blood. Results from allogeneic human serum have been mixed and
the drive for cord blood serum has been largely hinged on the primitive nature of the
source. Positive outcomes have been reported with autologous human serum. The
argument against the use of autologous serum so far is harvesting enough quantity and
the fact that serum from older patients may not support optimal expansion. These two
concerns can be simply addressed if people are encouraged to cryopreserve their
serum early enough. The major problem limiting the application of human serum is by
far the poor understanding of components in the serum that are inhibitory to the
expansion process. Serum contains growth factors and inhibitors, cytotoxic substances,
differentiation agents, attachments etc. Some of these components support a particular
cell type while they inhibit the growth of other cells. More knowledge of the cytotoxic
substances and other components that interfere with the growth factors and hormones
in the serum is crucial for future progress
Efforts should also be made to optimize the growth factors in autologous serum use for
expansion. Already, the process of serum collection has been manipulated to allow the
derivation of growth factors. An example of this manipulation is the derivation of PDGF
using cellaid. PDGF is a growth factor necessary for the stimulation and expansion of
early progenitors and committed progenitors cells. It is possible that more growth factors
could be sufficiently derived from autologous human serum without the need for
supplementation as more innovative manipulation strategies are explored.
Interestingly, more clinicians and biotechnology companies have begun to look in the
direction of autologous stem cell therapy. This renew interest has opened a new way in
which trial is being done to assess safety and efficacy of stem cell therapy outside the
traditional clinical trial rules. Examples of new ways in which an expansion protocol can
be assessed include patient sponsored studies where the patients pay the bill instead of
the government, open label study and institutional review board approval. This is the
right time to harness innovative approaches that will revolutionize stem cell therapy
globally.
Novel Elementary Strategy for Routine Clinical Application
One reason why stem cell therapy has been somewhat restricted to transplant referral
centers and some few stem cell treatment centers, stems from the fact that stem cell
transplantation where originally done with bone marrow cells. Despite the current reality
that PBSC can be easily harvested than bone marrow cells, our clinical, scientific and
regulatory authorities have sadly turned Luddite. Luddite is a term used to describe
group of English textile artisans who, beginning in the 19th century protested against,
and smashed, new labor saving machinery in the early industrial revolution, and now a
term for one opposed to new technology or new way of doing things.
It is now a standard of care to treat some selected patients with hematological disorder
with high-dose chemotherapy (HDC) using PBSC as support. Apart from cancer
treatment, stem cell therapy can also be used to regenerate damaged tissues and
organ including neurodegenerative diseases, spinal cord injury, heart disease etc. They
availability of stem cell therapy to practicing physicians will certainly benefit patients
who do not have access to this kind of treatment.
In this study, we use autologous serum harvested according to cellaid protocol and
supplemented with growth factors to expand PBSC in bench scale. Our result (table 2, 3
and 4) confirm the potential of autologous serum in the expansion of PBSC. It indicates
that bench scale expansion of PBSC is possible. To translate this success into the
clinic, the cells need to be expanded in a large scale. However, current efforts for large
scale clinical expansion have failed to continue at cell densities above 1.1 - 1.4 x 106
cell/ml. Some reasons suggested for this arrest includes; insufficient nutrients for the
cells in static culture and accumulation of metabolic products as a result of the
expansion. This condition might have produced the inhibitory effect in the culture
system which often promotes cell death. A fed batch system that continually increases
the volume of cultures in order to increase the space for the cells to expand optimally
and dilute away the inhibitory factors is a logical step to overcome the effect. This is
because the increasing culture volume of the fed batch strategy will eventually maintain
a lower cell density, thereby slowing the rate and impact of endogenous factor
accumulation. Incorporating automated fed batch into a system that allows continuous
observation through time lapse monitoring and a control of the physical and chemical
environment have not been adequately explored. Novel strategies incorporating this
should have a culture system where nutrient need, oxygen or pH profiles and culture
parameters known to have crucial influence over stem cell fate is rightly adjusted.
Exploring this strategy is extremely necessary for a successful clinical scale expansion
protocol.
Here, we present our novel clinical scale automated expansion system (Fig 11) which
consists of an optical fiber and sensor, pneumatic valves, cocktail media reservoir, filter
fluorescence spectrometer for optical biomass measurement, a CO2 incubator, time
lapse monitoring device, automated immune-staining device, a PH and temperature
controlling device. The culture setup is mounted inside a CO2 incubator and is
connected to an external valve which controls the flow of pressurized air from a
reservoir containing the cocktail media outside the incubator. The pressure component
was designed to force the cocktail media into a micro-valve pneumatic connection which
then supplies the media through a tube into a culture plate positioned inside an
incubator. Once the dose volume of the cocktail media per pump is known, the precise
flow rate can be established by defining the number of pumps per time unit. It has a
software program designed to signal and take measurement of scattered light through
the fiber optic from a fluorescence spectrophotometer. The scattered light of the culture
provides information regarding the accumulation of inhibitory factors and current nutrient
profile and is controlled by a motorized XY stage so that this information is available on
a fixed time. This software also regulates the CO2, O2 and temperature control box to
provide appropriate conditions inside the incubator through separate optic fibers. Based
on information analyzed by the software from the culture media in the incubating
system, a signal is sent to the valve to pump or halt the supply of nutrient from the
cocktail reservoir depending on the concentration of the inhibitory factors detected or
nutrients available. Time lapse monitoring device was fixed inside the incubating system
to provide a real time picture of the cultured cells so that a closed system is maintained
during culture and expansion. A tube is also positioned close to the culture plate and
directed outside the incubator so that occasionally a small volume of the cultured cell is
aspirated from the plate for automated immune-staining outside the incubator. This
function is also controlled by the software.
Stem cell expanded by our strategy can be translated into clinical settings by using
autologous human serum in preparing the cocktail media. A strategy like ours when fully
developed could encourage the use of autologous serum to expand PBSC in
community health center in accordance with good manufacturing practice.
CONCLUSION
In autologous stem cell therapy, patients donate their own blood cells and serum prior to
therapy and the expanded cells are infused back to the patients when it is needed. We
have demonstrated that autologous serum manipulated to derive PDGF can allow
bench scale expansion of PBSC, a lot more still need to be done to understand how
other growth factors and components that support expansion can be manipulated during
serum harvest. If autologous serum is sufficiently manipulated to derive other factors
that encourage expansion without the need for supplementation with recombinant
growth factors, it will further encourage the availability and reduce the cost of
autologous stem cell treatment in community healthcare centers. This is because all
that will be needed is the automated system that process and expand these cells, since
the serum and the cell to be expanded will be harvested from the patient.
ACKNOWLEDGEMENT
I sincerely thank my director of Admission and Student Affairs, Professor Doctor Piotr
Beck for supervising and advising me during this thesis.
My gratitude also goes to Dr Drake for his valuable comments during my studies.
A big thank you to the team at Nordica Fertility Centre Lagos, were this work was
carried out and to Androcare Laboratories and cryobank for providing the donor.
God bless you.
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peripheral blood stem cells. J Clin Oncol.1995; 13: 2547:2555.
24. Ketterer N, et al. Factors associated with successful mobilization of peripheral blood
progenitor cells in 200 patients with lymphoid malignancies. Br J Haematol. 1998; 103:
235:242.
25. Sato N, et al. In Vitro Expansion of Human Peripheral Blood CD34+ Cells.Blood.
1993; 82: 3600-3609.
26.Spinal cord injury. See July 14, 2004 Senate testimony by Dr. Jean Peduzzi-
Nelson.Available at:
http://commerce.senate.gov/hearings/testimony.cfm?id=1268&wit_id=3671.
27.Spinal cord injury .A more extensive testimony. Available at:
http://www.stemcellresearch.org/testimony/peduzzi-nelson.htm.
28.Spinal cord injury .Ms. Laura Dominguez. Available at:
http://commerce.senate.gov/hearings/testimony.cfm?id=1268&wit_id=3673.
29.Stilley CS et al. Changes in cognitive function after neuronal cell transplantation for
basal ganglia stroke, Neurology.2004; 63: 1320-1322.
30.Kondziolka D et al. Transplantation of cultured human neuronal cells for patients with
stroke.Neurology.2000; 55: 565-569.
31.Wollert KC et al. Intracoronary autologous bone-marrow cell transfer after myocardial
infarction: the BOOST randomised controlled clinical trial. Lancet.2004; 364: 141-148.
32.Britten MB et al. Infarct remodeling after intracoronary progenitor cell treatment in
patients with acute myocardial infarction. Circulation.2003; 108: 2212-2218.
33.Perin EC et al. Transendocardial, autologous bone marrow cell transplantation for
severe, chronic ischemic heart failure. Circulation.2003; 107: r75-r83.
34.Stamm C et al. Autologous bone-marrow stem-cell transplantation for myocardial
regeneration.Lancet.2003; 361: 45-46.
35.Tse H-F et al. Angiogenesis in ischaemic myocardium by intramyocardial autologous
bone marrow mononuclear cell implantation; Lancet.2003; 361: 47-49.
36.Love S et al. Glial cell line-derived neurotrophic factor induces neuronal sprouting in
human brain, Nature Medicine.2005; 11: 703-704.
37.Gill SS et al. Direct brain infusion of glial cell line-derived neurotrophic factor in
Parkinson disease. Nature Medicine.2003; 9: 589-595.
38. Pasquini MC, Wang Z. Current use and outcome of hematopoietic stem cell
transplantation: CIBMTR Summary Slides, 2013. Available at: http://www.cibmtr.org.
39. Diagram of Stirred bioreactor operated as a chemostat, with a continuous inflow
(feed) and outflow. Available at http://en.m.wikipedia.org/wiki/chemostat.
40.WAVE BioreactorTM 500/1000 system. Available at :
www.bioprocessonline.com/doc/wave-bioreactor-system-0002.
41.Bubble-Column and Air-Lift Bioreactor. Avalaible at
http://www.metal.ntua.gr/~pkousi/e-learning/bioreactors/page 11.htm.
42.Rotary Cell Culture System-motorized base with power supply and assorted vessels
Available at http://www.genengnews.com/gen-articles/rotating-bioreactors-for-
manufacturing/1899/.
43. Kim, SJ,et al. Microfluidic Automation Using Elastomeric Valves and Droplets:
Reducing Reliance on External Controllers. Small. 2012.
44. Ang M. Ex vivo expansion of hematopoietic stem cells: preclinical studies
and clinical application.Thesis, Univerisity of hongkong.2003.
Tables Table 1.Characteristics of Myeloid Cell Types
Adapted from (44)
Table 2.Percentage increase on seeded cell in culture
Table 3.Rate of CFU-GM Expansion
Table 4.Morphological changes in cultured myeloid cells
Day of
Culture
Initial Cell
Density.
(/ml)
Myeloblasts Promyelocytes Myelocytes/Metamyelocytes
0 5000/ml 36 ±6.2
2.8 ±1.4
1.3 ±0.4
7 5000/ml 8.9±0.3 14 ±3.9
22 ±4.8
12 5000/ml 1.2±0.7 27 ±5.3
41± 8.2
Table 5.Advantages and Disadvantages of Bioreactor Designs
Design Advantage Disadvantage
Stirred Culture Vessels Can operate in batch, fed-
batch, and perfusion and
can be adapted for
differentiation and/or
expansion
The hydrodynamic stress
promoted by stirring
Cell Culture Bags The come as disposable,
single-use, pre-sterilized
bioreactors. This make
them more convenient to
use
They can only be applied to
non-adherent stem cells or
those that grow as
aggregates or on
biocompatible micro-carrier
Bubble-Column and Air-Lift
Bioreactors
They permit high-efficiency
mass transfer with
excellent flow and mixing
properties
There is considerable back-
mixing between gas and
liquid phases, high
pressure drops, and bubble
coalescence
Rotary Systems They provide a well-mixed
environment for cell growth
There is limited control of
aggregate size and
nutrient/gas concentrations
as well as efficient gas
transfer through a silicon
membrane
throughout the vessel.
Necrotic centers can form,
leading to cell death inside
the aggregates.
Concentration gradients
resulting from mass
transfer limitations can
create uncontrolled
microenvironments
Microfluidic Culture
Systems
The microenvironment can
be controlled by adjusting
for example the perfusion
rate, providing a high-
throughput system for
evaluating the effects of
soluble factor concentration
gradients on different cell
processes
There may be high shear
stresses associated with
perfusion and the
continuous removal
of important molecules
secreted by cells
that could ultimately
compromise their
performance
Figures
Fig 1.Unrelated Donor Stem Cell Sources by Recipient Age Adapted from (38)
Fig 2.Photo of EasySep® Magnetic
Fig 3.Stirred bioreactor operated as a chemostat, with a continuous inflow (feed)and
outflow.The rate of medium inflow is controlled to keep the culture volume constant.
Adapted from (39)
Fig 4.GE Healthcare Life Science WAVE BioreactorTM 500/1000 system.
Adapted from (40)
Fig 5.Bubble column and Air-Lift Bioreactor. A)bubble column;b)air lift reactor;c)air lift
with particle separator; packed bed air lift.
Adapted from (41)
Fig 6.Rotary Cell Culture System-motorized base with power supply and assorted
vessels
Adapted from (42)
Fig 7.Microfluidic Bioreactor . Adapted from (43)
Fig 8.K System Lamina Air Flow Hood
Fig 9.G-185 Incubator K-System
Fig 10.Leica DM IRB inverted Microscope
Fig 11.A schematic diagram of our clinical scale expansion system for PBSC
ABBREVIATION BFU-E - Erythroid Burst-forming units
B M- Bone marrow
BSA -Bovine serum albumin
CAFC -Cobblestone area forming cell granulocyte-erythrocyte-macrophage
megakaryocyte colony
CFU-GM -Granulocyte-macrophage colony-forming units
CSF- Colony stimulating factors
DM -Defined media
DPBS- Dulbecco's Phosphate Buffered Saline
EPO -Erythropoietin
FACs -fluorescence-activated cell sorting
FBS -Fetal bovine serum
FCS -Fetal calf serum
FITC- Fluorescein isothiocyanate
G-CSF Granulocyte colony stimulating factor
GM -Granulocyte-macrophage
GM-CSF Granulocyte-macrophage colony-stimulating factor
GMP -Good manufacturing practices
HDC -High dose chemotherapy
HGF -Hematopoietic growth factors
HPC -Hematopoietic progenitor cells
PBSC-Peripheral Blood Stem Cell
DNA-Deoxyribonucleic Acid
HSC-Hematopoietic stem cell
BMT- Bone Marrow Transplant
PBSCT- Peripheral Blood Stem Cell Transplant
ANC-Absolute Neutrophil Count
CB- Cord Blood
IL-1b-Interleukin 1beta
IL-3-interleukin 3
IL-6- interleukin 6
SCF-stem cell factor
PDGF-Platelet Derived Growth Factor
TPO-Thrombopoietin
FGF-1 Fibroblast Growth Factor - 1
MSC-Mesenchymal Stem Cell
PGE2-Prostaglandin E2
HOXB4 �Homebox B4
SALL4-Sla like 4
EPO-Erythropoietin
HS-human serum
tPRP-thrombin-activated platelet releasate in plasma
pHPL- Pooled human platelet lysate
MGDF-megakaryocytes growth and differentiation factor
SCDF- stromal cell derived factor
PBMNC-Peripheral Blood Mononuclear Cell