1. Bio preservation of Red Cell Components 2. CULTURED RBCs 3. Solvent plasma Hypothermic storage Cryopreservation Lyophilization Desiccated RBCs Three major sources of cells are under consideration: circulating stem and progenitor cells from adults or from cord blood immortalized progenitors pluripotent stem cells. Immortalized Progenitors LIQUID CULTURE METHODS- by the SED (stem cell factor (SCF), erythropoietin, and dexamethasone) and STIF cocktails (stem cell factor, thombopoietin, insulin-like growth factor-2, fibroblast growth factor-2) ENUCLEATION- separation of extruded nuclei from cRBCs SCALING UP- using cord blood CD34+ cells in bioreactors and treated for 4 h with TNBP (tri-nitrobutylphosphate)solvent and with Triton X-100 detergent

1. 1. BIO PRESERVATION OF RED CELL COMPONENTS
2. CULTURED RBCS
3. SOLVENT PLASMA
Presented By- Dr. Shiny
Moderator- Dr. Nidhi Bansal

2. INTRODUCTION
 Biopreservation- Process of maintaining the integrity and
functionality of cells held outside the native environment for
extended storage times.
 The biopreservation of RBCs for clinical use can be categorized
based on the techniques used to achieve biologic stability and
ensure a viable state after long-term storage.

3. CURRENT RBC BIOPRESERVATION
APPROACHES
Hypothermic storage
Cryopreservation
Lyophilization

4. HYPOTHERMIC STORAGE
 Hypothermic storage (1–6°C) of RBCs became feasible nearly a
century ago, when Rous and Turner introduced the first glucose–citrate
storage solution.
 Detrimental changes in RBC metabolism and biological function
characterized with hypothermic storage. are commonly termed ‘the
hypothermic storage lesion’ (HSL)

5. RBC HSLs include -
 the depletion of vital metabolites, such as ATP, 2,3 diphosphoglycerate and
potassium
 cell senescence
 continuous decrease in pH
 loss of membrane components
 the release of microvesicles
 alterations in RBC rheological characteristics including filterability &
deformability

6. Possible outcomes include-
 impaired tissue oxygenation
 inflammation
 transfusion-related immunomodulation
 intravenous hemolysis

7. CRYOPRESERVATION
 Red cells can be frozen and are stable for prolonged periods in a frozen
state.
 Freezing of RBCs is mainly done to preserve units of blood with rare
phenotypes or to build blood inventory for emergency use in disasters.
 Done with cryoprotective agent- Glycerol
 Glycerol- After entering the red cell, it provides an osmotic force that will
stop water migration from the red cell due to extracellular ice formation.

8. Damage if frozen without cryoprotective agents-
• Red cell dehydration: When extracellular water freezes before intracellular water,
red cells collapse due to an osmotic pressure difference created between red cells and
their surroundings, resulting in the diffusion of intracellular water out of the red cells.
• Intracellular ice: When intracellular water freezes before extracellular water, red
cells rupture owing to an increase in intracellular salt concentration drawing in more
water and expanding the cell.

10.  Frozen RBCs can be stored for 10 years.
 Rare frozen units may be used beyond the expiration date as literature reports
satisfactory cell recovery and viability from units stored for up to 21 years.
 Deglycerolization- the unit is thawed at 37°C with gentle agitation taking
about 10 minutes for complete thawing. Glycerol is removed gradually from
thawed RBCs by washing with sterile saline solutions of decreasing
osmolarity to avoid red cell haemolysis

11. DESICCATED RBCS- LYOPHILIZATION
 Lyophilization (freeze-drying) involves the removal of most unbound
water from biologic materials through controlled freezing followed by
the sublimation of ice under vacuum.
 Effective lyophilization prevents sample shrinkage, minimizes
chemical changes, and maintains product solubility to allow easy
rehydration.

12.  Dry storage could potentially extend the product’s shelf life, reduce the
weight and volume of the storing vehicle, facilitate transportation and
save energy by eliminating the need for refrigeration.
 But unfortunately, the majority of the attempts to stabilize RBCs in the
dried state have resulted in poor cellular recovery, partially because of Hb
oxidation.
 At present, no reconstituted RBCs meet the standards for transfusion.

13. CONCLUSION
 Currently, the only way to successfully preserve Hb for transfusion medicine
applications is in its natural environment, the RBCs.
 Our ability, or inability, to preserve RBCs under various conditions (hypothermic,
frozen or dry) is largely dictated by the oxidative state of Hb.
 Our success in preserving RBCs through hypothermic or frozen storage relies, at least
in part, on keeping the Hb in its reduced state.
 Our failure to stabilize RBCs in the dried state or to create Hb-based oxygen
therapeutics has been affected by our inability to avoid Hb-induced oxidative damage.

14. Other NewerTypes of
Components

15. CULTURED RED BLOOD COMPONENTS
 In-vitro production of red cells
 Approximately 1% of the RBCs are eliminated from the circulation every
day, maintenance of the average adult RBC mass of 2.5 × 1013 requires the
daily production of more than 200 billion RBCs.
 HSCs sequentially differentiate into common myeloid progenitors and
megakaryocyte-erythroid progenitors and then into unipotent progenitors
restricted to the erythroid lineage.

16. SOURCES OF CULTURED RED CELLS
 Production of cRBCs for clinical purpose will require the identification of cell
sources that can reliably be tapped and used to develop a highly scalable production
procedure that recapitulates the erythropoiesis process
 Three major sources of cells are under consideration:
circulating stem and progenitor cells from adults or from cord blood
immortalized progenitors
pluripotent stem cells.

17. CIRCULATING STEM AND PROGENITOR CELLS
 With the best available methods, stem and progenitor cells found in 1 unit of cord blood can
theoretically be expanded into more than 500 units of RBCs
 This level of amplification is sufficient to significantly expand the blood supply of rare blood
groups, which would be an important milestone for the field, and justifies investment in this
technology.
 Drawback-
 their limited proliferation potential
 Since these cells are not immortal, production of cRBCs remains dependent on the donation-
based collection and testing system

18. IMMORTALIZED PROGENITORS
 In general, cellular immortalization requires simultaneous stimulation of proliferation and inhibition
of terminal differentiation and cell death
 Studies of murine erythroid precursors have led to the identification of several factors that control
these processes in erythroid cells. These factors include GATA-1, which promotes erythroid
development , and PU.1, which binds to GATA-1 and inhibits erythroid terminal differentiation
 Immortalization of erythroblasts can be achieved by manipulating the expression of transcription
factors, tumor suppressors, and nuclear receptors
 This approach might be safe because cRBCs are not oncogenic since they are enucleated and remain
fully functional after irradiation.

19. PLURIPOTENT STEM CELLS
 The third potential permanent source of cells for the production of cRBCs is
pluripotent stem cells, such as human embryonic stem cells (hESCs) and induced
pluripotent stem cells (iPSCs). The main advantages of these cells are that they are
immortal and karyotypically stable and that they can be reproducibly generated from
any individual with a variety of well-developed methods
 Genetic engineering of pluripotent stem cells, which is technically possible, further
expands the potential use of these cells as a source of cRBCs.

20. METHODS
 LIQUID CULTURE METHODS- by the SED (stem cell factor (SCF),
erythropoietin, and dexamethasone) and STIF cocktails (stem cell factor,
thombopoietin, insulin-like growth factor-2, fibroblast growth factor-2)
 ENUCLEATION- separation of extruded nuclei from cRBCs
 SCALING UP- using cord blood CD34+ cells in bioreactors

21. SOLVENT-DETERGENT FFP
 The solvent/detergent treatment is an established virus inactivation technology that
has been industrially applied for manufacturing plasma derived medicinal products for
almost 30 years.
 Solvent/detergent plasma is a pharmaceutical product with standardised content of
clotting factors, devoid of antibodies implicated in transfusion-related acute lung injury
pathogenesis, and with a very high level of decontamination from transfusion-
transmissible infectious agents.
 The solvent/detergent (S/D) pathogen inactivation method was developed in the early
eighties for the inactivation of enveloped viruses in plasma-derived medicinal products

22. METHOD
1. Filtration with a 1-μm filter to remove cells and debris
2. FFP is thawed rapidly and treated for 4 h with TNBP (tri-
nitrobutylphosphate)solvent and with Triton X-100 detergent, both at 1 %
3. TNBP is then removed by extraction with castor (or soybean) oil and the Triton
X-100 by hydrophobic chromatography
4. These processes are followed by sterile filtration with a 0.2-μm filter and
aseptic packaging into the final product

23. ADVANTAGES OF SOLVENT-
DETERGENTTREATMENT OF FFPS

24. 1. VIRAL AND BACTERIAL DECONTAMINATION
 Decontamination of S/D plasma is guaranteed
 Immunological neutralisation
 Double filtration removes cells, cell fragments, and bacteria
 Highly effective in disrupting lipid membranes (lipid envelop viruses)
 Extensive post-marketing clinical experience have clearly shown that the S/D
treatment achieves a rapid, irreversible, and thorough inactivation of enveloped
viruses
 Although bacteria rarely contaminate FFP because of its storage conditions, it
inactivates some gram-positive bacteria

25. 2. STANDARDISATION & DILUTION EFFECT
 The dilution and neutralisation of antibodies and allergens during the
industrial process of S/D plasma pooling can reduce the incidence of
allergic reactions in recipients
 Neither anti-human leucocyte antigen (HLA) nor anti-human neutrophil
antigen (HNA) antibodies are detectable in S/D plasma as they are diluted
and neutralised by the presence of leukocytes or their fragments, which are
then removed by the S/D treatment- For this reason countries using S/D
FFP have not reported any transfusion-related acute lung injury (TRALI)
case due to the transfusion of plasma

26. 3. CLINICAL EXPERIENCE
 The high level of safety towards allergic reactions, TRALI and risk of
transmission of enveloped viruses and of final standardization of coagulation
factor content has recently led to the wide use of S/D plasma
 A number of clinical studies have analysed in the last two decades the
efficacy and safety of this biological agent in various clinical settings,
including congenital coagulation deficiencies, acquired coagulopathy of liver
disease, coumarin-reversal and surgery

27. REFERENCES
 Biopreservation of red blood cells – the struggle with hemoglobin oxidation- The FEBS Journal- VOL 277
Issue 2
 Biopreservation of Red Blood Cells: Past, Present, and Future Transfusion Medicine Reviews Volume 19,
Issue 2
 DJHS Transfusion Medicine Technical Manual Volume 3
 Concise Review: Production of Cultured Red Blood Cells from Stem Cells byEric E. Bouhassira- Stem cell
translational medicine
 Solvent/detergent plasma: pharmaceutical characteristics and clinical experience