1 IntroductIon

Erythropoietin (EPO), a member of the type I cytokine super-family, was first

identified as the hormone that stimulates erythroid progenitors within the bone marrow

to mature into erythrocytes. Erythropoietin is also involved in the control of hemoglobin

synthesis and red blood cell concentration in blood. However, in the recent past, many

other physiologic roles have been attributed to EPO (Erbayraktar et al., 2003), such as (i)

EPO is now known to be a local product of diverse cells that specifically protect cells

from potential cytotoxic events and (ii) EPO maintains and protects tissue function,

especially during metabolic stress. Tissue oxygen tension is one of the important factors

that regulate the production of EPO in in vivo condition (Fisher et al., 1996).

Erythropoietin is one of the few hematopoietic growth factors which behave like

a hormone. It is a member of the hematopoietic growth factor and its principal function

is to couple oxygen delivery by circulating red cells (Cotes, 1994) to long-term tissue

oxygen needs. Even though EPO is produced primarily in the kidneys, it is also produced

in the liver to a small extent in adults. EPO interacts in the bone marrow with specific

receptors expressed on the surface of erythroid progenitor cells to initiate their entry into

cell cycle if dormant or to maintain their viability while differentiating, if they are

already in active cell cycle. Erythropoietin achieves its effects by causing homo-

dimerization of its receptor with the resultant auto-phosphorylation of the tyrosine kinase

JAK2 (Kitamura et al., 1991 and Livnah et al., 1998) and phosphorylation of the

receptor itself, as well as various substrate proteins leading to the up regulation of a

number of signaling pathways and the activation of gene transcription

(http://www.bioxys.com/i_Gentaur/epo.htm).

Erythropoietin (EPO) stimulates the proliferation and differentiation of erythroid

cells leading to an increase in the number of reticulocytes in the blood, which

consequently increases the blood’s haematocrit and haemoglobin concentration (Hitomi

et al., 1988). EPO exerts its activity by binding to specific cell surface receptors on

erythroid progenitor cells. This hormone (EPO) is produced mainly by the kidneys

(primarily by cells of the peri-tubular capillary endothelium) in human adults (Jacobson

et al., 1957 and Jelkmann, 1986).

In addition to the kidney, EPO is also produced locally by other tissues in a

paracrine–autocrine manner (Klein et al., 1989). Clinically, it has been observed that,

anemia associated with renal failure often results from a decreased level of EPO

(Coleman and Brines, 2004). During severe illness, erythropoiesis usually fails because

of a blunting of the kidney-erythropoietin (EPO)-bone marrow axis. In this condition,

clinical studies have shown that recombinant human erythropoietin (rhEPO),

administered in pharmacological amounts, reduces the need for blood transfusions

significantly (http://www.caspases.org/showabstract.php?pmid=15469595). Plasma

levels of EPO can increase by one hundred fold in severe anemic condition, due to

insufficient oxygen supply to the tissue, while its concentration can plummet as a result

of renal dysfunction.

EPO Receptor and the Cytokine Receptor family

The gene for EPO receptor was cloned by Noguchi et al., (1991) from murine

erythroleukemia cells and it became clear that the EPO receptor belongs to the cytokine

receptor family that comprises receptors for the various interleukins (IL’s) namely, GM-

CSF, Granulocyte Colony-Stimulating Factor (G-CSF), Growth hormone and Prolactin

(Yoshimura and Arai, 1996). The special characteristic of cytokine receptor family is that

they are switched on i.e., up on activation, the receptor transduce signals to the interior of

the cell by the formation of homo- or hetero-oligomers (dimers or trimers) (Sasaki et al.,

1987 and Sawyer et al., 1989).

On binding of EPO to its receptor, results in homo-dimerisation of the receptor

leading to the activation of several signal transduction pathways- JAK 2/STAT5 system,

G-protein (RAS), calcium channel and kinases (Tilbrook and Klinken,1993; Wen et al.,

1994) (Figure 1). Cytokines such as the interleukins create redundancy through the

common use of a single receptor subunit as they can affect a relatively wide range of

cells and have redundant biological activity. Whereas, on the other hand, EPO and G-

CSF act with high specificity on a relatively limited range of cells, so it was probably

unnecessary for their receptors to share one of the subunits.

EPO Receptor and Signaling Pathway

It is thought that the signal for cellular proliferation and differentiation into

erythroblasts originate at the EPO receptor. The EPO receptor’s cytoplasmic domain can

be divided into two major regions. Roughly half of the cytoplasmic domain, the part

lying near to the plasma membrane, is required for generating the signals for proliferation

Figure 1: The core module of the JAK-STAT signaling pathway, wherein

extra-cellular binding of EPO to its receptor phosphorylates JAK2 protein

intra-cellularly that in turn activate STAT5 homo-dimerisation, which

recognizes nuclear localization sequence on the target gene and cause

proliferation (http://www.pnas.org/content/100/3/1028/F1.expansion.html)

and differentiation such as the induction of globin synthesis and the remaining

half is not required for this signaling, and, conversely, it acts to dampen the signals. It is

known that a tyrosine kinase (Ohashi et al., 1994) called JAK2 associates with the region

near the plasma membrane undergoes autophosphorylation (Witthuhn et al., 1993). Auto-

phosphotylation of JAK2 (Yoshimura et al., 1996) stimulates the phosphorylation of

STAT, a transcription factor, which phosphorylates the EPO receptor (Yoshimura and

Arai, 1996)

It is thought that JAK2 plays a significant role in promoting cellular proliferation

and the STAT is activated by the phosphorylation and it then translocates to the nucleus,

recognizes a specific base sequence called gama activated site (GAS) in the promoter

region of its target gene, and initiates transcription. At present, it is known, that the

STAT whose activation is mediated by the EPO receptor is STAT5, and the target genes

are CIS which has a src homology 2 (SH2) domain (Yoshimura et al., 1995) a molecular

structure that recognizes a phosphorylated tyrosine) and oncostatin M (OSM) which is a

pleiotropic cytokine. However, STAT5 activation and the target genes are not unique to

the EPO receptor, and they also occur with the IL-2 and IL-3 (Tadmori et al., 1989)

receptors. Moreover, the JAK2 substrate which is directly linked to cellular proliferation

is still unknown and at present, studies are under way to determine the transcription

factors specific to EPO and their target genes, as well as the substrates of JAK2

(Yoshimura and Arai, 1996).

EPO Receptor Phosphorylation and Cessation of the Signal

On the other hand, it is necessary for the receptor to undergo tyrosine

phosphorylation at the cytoplasmic tail region far from the plasma membrane. The signal

transduction pathway that originates by this phosphorylated tyrosine and is mediated by

proteins with SH2 domains becomes activated. First, a GTP/GDP exchange factor called

SOS, which is mediated by SHC and growth factor receptor-bound protein 2 (Grb2),

converts a RAS protein to its GTP form after migrating to the plasma membrane. Raf-

MAP kinase kinase-MAP kinase cascade is activated by RAS protein, and ultimately

initiates the transcription of oncogenes such as c-fos and c-jun (Maruyama et al., 1994).

An enzyme called phosphoinositide 3 (PI 3) kinase that binds to the tyrosine

phosphorylation site of the receptor results in the formation of a second messenger. In

certain types of fibroblasts, it is known that this pathway is a requirement for DNA

synthesis and these signal transduction pathways are not unique to the EPO receptor, and

they are also activated by most growth factor receptors, so they are not necessarily

required for EPO-induced proliferation (Fraser et al., 1989). The SH-PTP1 (tyrosine

phosphatases), also called HCP has an SH2 domain and is specific to blood cells

associates with the tyrosine phosphorylation site of the receptor and promotes the

dephosphorylation of JAK2 and in other words, the role of SH-PTP1 is to stop generation

of the signal (Klingmuller et al., 1995).

In mutations the receptors do not undergo tyrosine phosphorylation, JAK2

activation continues for a longer period of time, and thus the signal is generated more

efficiently as cytoplasmic tail region of the receptor will be lacking far from the plasma

membrane (Genc et al., 2004). In fact, a mutation was discovered in one patient with a

mild case of familial erythrocytosis, in which the C-terminus of the EPO receptor was

missing 70 amino acids (Chapelle et al., 1993). The patient’s erythroblasts showed an

increased sensitivity to EPO and this was a dominant genetic trait and in this family the

impairment was not severe enough to be called an illness, and in fact it is said that this

patient was proficient enough athletically to compete for a gold medal at the Olympics.

The reason that athletes undergo training at high altitudes more specifically is to boost

EPO production because of the lower oxygen partial pressure, and this brings about the

desired effect of sustained athletic capability due to a resultant increase in red blood cells

(Wide et al., 1989). However, the same effect had occurred naturally in this athlete due to

accelerated receptor capability (Yoshimura and Arai, 1996).

The process of erythropoiesis is regulated by a feedback loop involving

erythropoietin (Macdougall, 2000) (Figure 2) so that, the production of red blood cells is

equal to the destruction of red blood cells and the red blood cell number is sufficient to

sustain adequate tissue oxygen levels but not so high as to cause sludging, thrombosis, or

stroke in non-disease states (Ohlsson and Aher, 2006). In response to low oxygen levels,

Erythropoietin is produced in the kidney and liver. This happens as erythropoietin is

bound by circulating red blood cells and low circulating numbers lead to a relatively high

level of unbound erythropoietin, which stimulates production in the bone marrow

(Drueke et al., 2006).

Figure 2: Erythropoiesis pathway along with different blood cell formation from

common source, Hemocytoblast. (http://nursingcrib.com/case-study/leukemia-case-study)

History

In 1906, Paul Carnot and his assistant De flandre proposed the idea that

hormones regulate the production of red blood cells (RBC). After conducting

experiments on rabbits subject to bloodletting, Carnot and Deflandre attributed an

increase in red blood cells in rabbit subjects to a hemotopic factor called hemopoietin.

Paul Carnot and C. Deflandre described the existence of a hormone responsible for

erythropoiesis. They have observed that regeneration of blood after bloodletting is under

the influence of a humoral process (a process controlled by a substance in the blood) and

termed this substance the generic name “Hemopoietine”

(http://www.hematology.org/Publications/50-Years-in-Hematology/4740.aspx). In 1948,

Eva Bonsdorff and Eeva Jalavisto continued to study red cell production and later called

the hemopoietic substance ‘Erythropoietin’ (EPO). Further studies by Reissman and

Erslev in the year 1950 investigating the existence of EPO, demonstrated that a certain

substance circulated in the blood is able to stimulate red blood cell production and

increase hematocrit and this substance was finally purified and confirmed as

Erythropoietin, opening doors to therapeutic uses for EPO in diseases like anemia

(Jelkmann, 2007).

In 1970, Haematologist, John Adamson and nephrologist, Joseph looked at

various forms of renal failure and the role of the natural hormone EPO in the formation

of red blood cells. In the 1970’s studying sheep and other animals, the two scientists

helped establish that EPO stimulates the production of red cells in bone marrow and

could lead to a treatment for anemia in humans. Goldwasser and Kung began work to

purify human EPO in 1968, managed to purify in mg by 1977 and the pure EPO allowed

the amino acid sequence to be partially identified and the gene to be isolated (Jelkmann,

2007). Later, a NIH-funded research program at Columbia University led to the synthesis

of EPO. Columbia University patented the technique and licensed it to an US based

company, Amgen (http://www.tititudorancea.com/z/erythropoietin.htm).

In the 1980s, Adamson, Joseph, Joan, Michael and Jeffrey conducted a clinical

trial at the Northwest Kidney Centers for a synthetic form of the hormone, Epogen

produced by Amgen. The trial was successful, and the results were published in January

1987 in the New England Journal of Medicine (Eschbach et al., 1987).

The human erythropoietin gene was isolated by Lin et al., in 1985 from a

genomic phage library. They were able to characterize it for research and production and

their research demonstrated that the gene for erythropoietin encoded the production of

EPO in mammalian cells that was biologically active in vitro and in vivo (Vogt et al.,

1989). This had further opened up the door for the industrial production of recombinant

erythropoietin (rhEPO) as a therapeutic protein for treating anemia patients (Lin et al.,

1985).

Biochemical and Structural Aspects of EPO

Erythropoietin is a monomeric sialo-glycoprotein with a molecular mass of 30.4

kda, containing 165 amino acids linked to four carbohydrate chains that incorporate

sialic acid residues.

The protein is heavily glycosylated (three N-linked glycosylation sites at Asn -

24, 38 and 83, and one O-linked glycosylated site at Ser -126), and the carbohydrate

chains corresponds to 40 % of the EPO molecular mass. The polypeptide chain of EPO

contains two intra-molecular disulfide bonds in at Cys-7 to Cys-161 and at Cys-29 to

Cys-33 positions (Figure 3).

The human erythropoietin gene is present on chromosome # 7 (q11-22) and is

spanned by five exons and four introns, which produce a post-transcriptional single

polypeptide containing 193 amino acids. The final gene product is devoid of 27 amino

acid long hydrophobic secretory sequence. Based on circular dichorism (CD) spectral

analysis of EPO, it has been suggests that its secondary structure contains 50 % of helix

moiety; with spatial arrangement of two helical pairs running anti-parallel similar to the

one seen in growth hormone (Lai et al., 1986; Jelkmann, 1992 and Inoue et al., 1995).

Like several glycoprotein hormones, EPO also exists as a mixture of isoforms.

This micro-heterogeneity is related to the charged carbohydrate moiety of the protein and

is associated with the presence or absence of terminal N-acetyl neuraminic acid residues

with varying amounts of acetylation and the presence or absence of N-acetyl lactosamine

extensions. Therefore, the degree of sialylation of polysaccharide chains strongly

influences the electrophoretic mobility and iso-electric point of the molecule (Lai et al.,

1986 and Jelkmann, 1992).

Figure 3: Erythropoietin Structure with 3 N-linked glycan structures at Aspargine

24, 38 & 83 and O-linked glycan at Serine 126 (http://www.steroidreport.com/2008/07/21/biosimilar-epo-agents)

Recombinant Human Erythropoietin (rhEPO)

In 1970’s human EPO was first isolated from urine and later purified and interest

in developing clinical uses for EPO led to the discovery of the gene encoding EPO, and

several groups devised recombinant DNA methods to produce EPO by the mid-1980s

((Lin et al., 1985 and Tabbara, 1993). Later, rhEPO was produced by culturing

genetically modified Chinese hamster ovary (CHO) cells. The gene of rhEPO was cloned

and inserted into a eukaryotic expression vector. CHO cells were transfected with this

vector carrying the human EPO gene (Law et al., 1986) and further selection were

performed to select a clone for its high expression level and growth capacity. The N-

glycosylated moiety of recombinant human erythropoietin has three main functional

units; the main core, the branched portion and the terminal component with each unit

having a specific role. However, the function of the O-glycosylated unit, a component

constituting about 3 % of the total mass of recombinant human erythropoietin, remains to

be defined (Ng et al., 2003).

Action of Erythropoietin

Erythropoietin promotes neuronal survival after hypoxia and other metabolic

insults by largely unknown mechanisms. Whereas, apoptosis and necrosis have been

proposed as mechanisms of cellular demise, and either could be the target of actions of

EPO (Siren et al., 2001).

Human bone marrow cells form small erythroblast colonies in five to seven days

when they are cultured in a semisolid medium containing EPO and by day 10 large

erythroblast colonies appear that resemble fireworks ("burst" colonies). Colony forming

units-erythroid (CFU-E) or late-stage erythroblast progenitor cells are the original cells in

the former colonies where as in the latter colonies they are called burst forming units-

erythroid (BFU-E) or early-stage erythroblast progenitor cells. Red blood cells are

produced during the process of differentiation of stem cells to burst forming units-

erythroid (BFU-E), colony forming units-erythroid (CFU-E) and erythroblasts (Figure 4).

CFU-E cells show greater sensitivity to EPO even though EPO acts on both BFU-E and

CFU-E cells. Other factors such as Stem Cell Factor (SCF), IL-3, IL-4, and granulocyte

macrophage colony-stimulating factor (GM-CSF) must be present together with EPO for

BFU-E cell proliferation and in erythroblasts beyond the CFU-E stage, sensitivity to EPO

Figure 4: Role of Erythropoietin at different stages of Erythrocyte formation (http://www.wjgnet.com/1007-9327/full/v15/i37/WJG-15-4617-g001.jpg)

decreases as the cells mature (http://www.wjgnet.com/1007-

9327/full/v15/i37/WJG-15-4617-g001.jpg).

Recombinant Human Erythropoietin as Therapeutic Protein

Recombinant human erythropoietin has been found to be efficacious in the

treatment of EPO-deficient anemia during illness. Clinical studies have shown that

(rhEPO), administered in pharmacological doses, significantly reduces the need for

blood transfusions. Here, EPO rescues cells from apoptosis which is similar to its role in

the bone marrow and additionally, EPO reduces inflammatory responses, restores

vascular auto-regulation, and promotes healing. The results obtained from many studies

(including a phase II clinical trial in ischemic stroke) demonstrate that rhEPO protects

the brain, spinal cord, retina, heart, and kidney from ischemic and other types of injury

(Perez-Oliva et al., 2005).

EPO is the main regulator of the production of red blood cells (Fandrey, 2004)

and it is principally involved in the recruitment and differentiation of erythroid

progenitor cells and aids in their maintenance and survival. Erythropoietin also

stimulates the synthesis of hemoglobin during hypoxia (Ratcliffe, 2003). In the last 15

years, the ready availability of recombinant human erythropoietin (rhEPO, Epoetin alfa)

has permitted the clinical investigation and application of this hormone to the treatment

of anemia in various patient populations. Epoetin alfa has been shown to accelerate

erythropoiesis and reduce allogeneic blood transfusion in major elective, non-cardiac,

non-vascular surgery and in certain anemic patients (Aher and Ohlsson, 2006) with

chronic renal failure, non-myeloid malignancies and in human immunodeficiency virus

infection. In addition to improving hematologic parameters, Epoetin alfa therapy can

enhance health-related quality of life in these patients. The success of Epoetin alfa (EPO)

in treating anemia in other surgical populations suggests that it may be of benefit in

treating the peri-operative anemia that is highly prevalent in patients who have

undergone gynecologic surgery (Bieber, 2001).

Recombinant human erythropoietin (rhEPO) has been shown to increase survival,

decrease hospitalizations, improve brain and cognitive function and improve quality of

life for renal patients. RhEPO has revolutionized the treatment of anemia of chronic

renal failure and much has been learned about the normal and pathologic physiology of

anemia because rhEPO has become available to investigators, and this has been widely

applied (Tong and Nissenson, 2001).

Industrial production of rhEPO & Applications

Erythropoietin produced by recombinant DNA technology in mammalian cell

culture is available as a therapeutic agent and is used in treating anaemia resulting from

chronic kidney disease and myelodysplasia, from the treatment of cancer (chemotherapy

and radiation), and from other critical illnesses (heart failure). Recombinant human

erythropoietin is available in several forms in the market as biomedicine namely, Eprex,

Epogen, Epokine, Epotin, Betapoietin, Relipoietin, Methoxy Polyethylene Glycol-

Epoetin Beta, Darbepoetin etc (http://www.steroidreport.com/2008/07/21/biosimilar-

epo-agents). The main applications of rhEPO as a therapeutic agent are as follows:

Anemia due to chronic kidney disease and Lung disease (Macdougall et

al., 1996 and Miller et al., 1981)

Anemia due to treatment for cancer (Schrijvers et al., 2010 and Buemi et

al., 2005)

Anemia in critically ill patients (Corwin et al., 2007)

Treatment of acute ischemic stroke (http://books.google.com.au

/books?id=A76u7g0QnskC)

Retinal survival factor (Becerra and Amaral, 2002)

Neuroprotection (Ehrenreich et al., 2004 and Juul, 2004)

Wound healing (Haroon et al., 2003)

Therapeutic demand for recombinant EPO quickly made it to market as a

therapeutic protein produced by many pharmaceutical industries, world over, for the

treatment of anemia resulting from a host of conditions, primarily kidney failure, HIV

infection in patients treated with AZT (Azidothymidine), and anti-cancer

chemotherapeutic drugs (Schellekens and Ryff, 2002). In the industrial production

conditions, the recombinant CHO cells are grown in roller bottles containing serum free

medium. Recombinant human EPO synthesized by recombinant CHO cells is secreted

into the culture medium from where it is purified by means of liquid chromatographic

methods. The rhEPO so obtained has similar biological and chemical characteristics like

that of international reference standard of EPO. Most of the pharmaceutical industries

have a similar strategy for the production of rhEPO, worldwide (Table 1).

The strategy adopted for the production of a recombinant CHO cell line secreting

human erythropoietin (rhEPO) includes,

A. Human EPO gene isolation and cloning

Preparation of genomic DNA

PCR gene amplification

Cloning and sequencing

Cloning in expression vector.

Transfection of CHO DHFR-cells and amplification with

methotrexate (MTX).

The human EPO gene was obtained from genomic human DNA by PCR

(Polymerase Chain Reaction) amplification. It was cloned in a plasmid vector for its

sequencing, sub-cloned in a eukaryotic expression vector, co-transfected with another

plasmid containing the DHFR gene, in Chinese Hamster Ovary cells (CHO). Genetic

amplification was made using MTX and the EPO producing cell clones were selected by

ELISA test. Total RNA was isolated from EPO producing cells and amplified by PCR.

The amplified product was cloned and sequenced to compare the isolated gene with that

of the EPO gene sequence documented in Gene Bank.

Table 1: Some of the major rhEPO producing companies

(http://www.steroidreport.com/2008/07/21/biosimilar-epo-agents)

Brand name Company

Epogen Amgen, California, USA

Procit Amgen, California, USA

Eprex Cilag, Schaffhausen, Switzerland

Neo recomon Roche, Basel, Switzerland

Ceriton Ranbaxy, Newjersy, USA

Epofit Intas Biopharmaceuticals Ltd., Ahmedabad, India

Erykine Intas Biopharmaceuticals Ltd., Ahmedabad, India

Hemax Hindustan antibiotics, Pune, India

Shanpoietin Shanta Biotechnics, Hyderabad, India

Wepox Wockhardt, Mumbai, India

Zyrop Zydus Biogen, Ahmedabad, India

CHO cell line mutated to be deficient in the DHFR gene (CHO- DHFR) in order

to facilitate genetic amplification with MTX. The cells were co-transfected, by the

calcium phosphate technique (Jordan et al., 1996).

B. Isolation of rhEPO- producing CHO cells

Clones that grew in presence of MTX were isolated, amplified in fresh medium

and once grown; the culture supernatant was tested to measure the rhEPO secretion using

a commercial ELISA test kit (R&D Systems Inc., MN, USA).

C. Control of the EPO mRNA produced by the rhEPO-CHO cells

Preparation of cell RNA

Preparation of specific DNA:

Amplification and cloning of EPO cDNA

D. Sequencing of the EPO cDNA cloned in pUC18 vector

The sequence obtained by amplification of the human EPO cDNA needs to be

identical to the published sequence of human EPO (http://www.ncbi.nlm.nih.gov/

nuccore/182197?report=fasta Gene Bank: Accession No.M11319).

There is a large gap in the demand (for therapy) and supply (production) of

rhEPO as a therapeutic protein. It is estimated that, even if rhEPO is produced

continuously over the next one decade, it may not fulfill the market demand (Figure 5).

One of the foremost reasons that limit the production of rhEPO to cater to the

market demand is the use of adherent cells for the production. This process requires more

man-power and adequate space. Further, automation is also very difficult to be achieved

when adherent cells are used in industrial production. This procedure requires solid

substrate for attachment and trypsinization for dislodging the cells. In addition, cells

cannot be aliquoted often, for continuous monitoring of cell viability, morphology and

contamination.

Recombinant human EPO production (small or large scale) using adherent cells

require roller bottles along with the specially designed bottle holder and incubator. Due

to the restrictive nature of adherent cell culture, the mode of production is hampered by

low cell-specific productivity. The large-scale production can be achieved by the

adaptation of adherent cell lines to suspension type culture (Ghani et al., 2006).

Figure 5: Market scenario for rhEPO showing the constant demand for EPO

(http://www.nature.com/nbt/journal/v28/n11/images_article/nbt1110-1165-F2.gif)

Other disadvantages of adherent cell cultures include;

• Loss of cells, indirectly the amount of product due to poor trypsinization.

• Cell death, loss of cell properties and cell leakage due to over trypsinization.

• Highly skilled/trained man-power is required during the course of production.

• Automation is highly impossible.

There are only two alternatives to overcome these limitations in using the

adherent cells. One is to clone gene of interest directly in suspension cells and the other

is to adapt the existing adherent cells to suspension form.

Suspension cells have following added advantages over the adherent cells (Birch

and Arathoon, 1990).

Require less space and

Less equipment.

Material can be reused viz., Conical flask , Spinner flask , Bioreactor

(Figure 6) etc.

Can reduce labour by 90 %.

Can be automated easily.

However, it is pertinent to note that not all cell lines can be adapted to suspension

growth. It should also be note that in general, normal diploid anchorage-dependent (must

be attached to a substrate to grow) cells cannot be adapted without the use of micro-

carrier beads to which they can attach (http://atcc.custhelp.com/app/answers

/detail/a_id/40).

Use of specially modified suspension culture medium and patience, is the key for

adapting cells to suspension growth from adherent type. Cultures are usually grown in a

small 250 mL to 1,000 mL spinner cultures (with half the volume of actual medium), in

glass vessels with a stirring paddle suspended inside and is driven at 50 to 100 rpm by a

magnetic stirrer. The spinner with suspension culture medium should limit the

concentration of calcium and magnesium ions to a level that prevents clumping/clogging

of cells. Further, use of serum in the medium requires addition of anti-foaming agent

(http://atcc.custhelp.com/app/answers/detail/a_id/40).

Figure 6: Bioreactor/ Fermentor

(http://www.php.wur.nl/NR/rdonlyres/9717762E-3A59-41CA-9488-

E198F28EE262/11543/fermentor2.jpg)

The glass culture vessel is usually siliconized (coated as a very thin film) so as to

prevent the cells from sticking to the glass. Initially, most of the cells tend to plate out on

the glass surface of the vessel or form large clumps with each other. After each passage,

those cells that are in suspension are used as inoculum’s to seed next set of containers.

As a function of time, the populations of cells that have adapted to suspension form,

wherein they show non-self aggregation or adhere to glass surface as readily as the

parental line are selected. Possibly, these trained cell line may have lost or acquired

characteristics that are independent and different from that of the original cell population

(http://atcc.custhelp.com/app/answers/detail/a_id/40).

Alternatively, adherent cells can also be adapted in conical flasks, after cells are

passaged several times in T-flasks so as to expand the quantity of cells. Later, they are

seeded into conical flasks (125 mL) at 5 105 cells/mL in a total volume of 30 mL

growth media. All flasks are placed on an orbital shaker (~120 rpm) and maintained at a

temperature of 37oC in a humidified incubator with 5 % CO2. Once the cells are adapted

to grow as suspension culture, it is possible to scale up the cultures in spinner flasks or

bioreactors (http://tools.invitrogen.com/content/sfs/manuals/3919.pdf).

Furthermore, advancement in technology has led to the development of dedicated

cell adaptation systems. This system includes a dedicated CO2 incubator and a shaker

(Figure 7) for adapting adherent cell lines to suspension ones. To avoid the use of micro-

carrier beads for growing adherent cells, they are normally adapted in an incubator using

a combination of mechanical and chemical means (www.ls.manchester.ac.uk /research/

facilities/Fermentation/equipment/celladaptation).

Figure 7: Dedicated cell adaptation system with CO2 incubator & orbital shaker

(www.ls.manchester.ac.uk/research/facilities/Fermentation/equipment/celladaptation)