Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy

Подождите немного. Документ загружается.

Grifﬁths JB (1988), Cell products: an overview, In: Spier RE, Grifﬁths JB (Eds),

Animal Cell Biotechnology, Vol. 3, Academic Press, London, pp. 179–221.

Hayﬂick L, Moorhead PS (1961), The serial cultivation of human diploid cell strains,

Exp. Cell Res. 25:585–621.

Heath CA (2000), Cells for tissue engineering, Trends Biotechnol. 18:17–19.

Hot Drugs (2004), Nat. Rev. Drug Discov., Suppl. Vol. 3.

Hwang WS, Ryu YJ, Park JH, Park ES, Lee E, Koo, JM, Jeon HY, Lee BC, Kang SK,

Kim SJ, Ahn C, Hwang JH, Park KY, Cibelli JB, Moon SY (2004), Evidence of a

pluripotent human embryonic stem cell line derived from a cloned blastocyst,

Science 303:1669–1674.

Kohler G, Milstein C (1975), Continuous cultures of fused cells secreting antibody of

predeﬁned speciﬁcity, Nature 256:495–497.

Laﬂamme MA, Murry CE (2005), Regenerating the heart, Nat. Biotechnol. 23:

845–856.

Land H, Parada LF, Weinberg RA (1983), Tumorigenic conversion of primary

ﬁbroblasts requires at least two co-operating oncogenes, Nature 304:596–602.

MacDonald C (1990), Development of new cell lines for animal cell biotechnology,

CRC Crit. Rev. Biotechnol. 10:155–178.

Mayhall EA, Paffett-Lugassy N, Zon LI (2004), The clinical potential of stem cells,

Curr. Opin. Cell Biol. 16:713–720.

Merten O-W, Cruz PE, Rochette C, Geny-Fiamma C, Bouquet C, Gonc¸alves D,

Danos O, Carrondo MJT (2001), Comparison of different bioreactor systems for

the production of high titer retroviral vectors, Biotechnol. Prog. 17:326–335.

Monoclonal antibodies and therapies (2004), Supplement to Nature Publishing Group,

http://www.nature.com/focus/antibodies/index.html, accessed in February 2006.

Noguchi M, Yi H, Rosenblatt HM, Filipovich AH, Adelstein S, Modi WS, McBridge

OW, Leonard WJ (1993), Interleukin-2 receptor gamma chain mutation results in

X-linked severe combined immunodeﬁciency in humans, Cell 73:147–157.

Powers MJ, Janigian DM, Wack KE, Baker CS, Beer Stolz D, Grifﬁth LG (2002),

Functional behavior of primary rat liver cells in a three-dimensional perfused

microarray bioreactor, Tissue Eng. 8:499–513.

Ruley HE (1983), Adenovirus early region 1A enables viral and cellular genes to

transform primary cell in culture, Nature 304:602–606.

Shah AC, Benos D, Gillespie GY, Markert JM (2003), Oncolytic viruses: clinical

applications as vectors for the treatment of malignant gliomas, J. Neurooncol.

65:203–226.

Spier RE (2000), History of animal cell technology, In: Spier RE (Ed.), Encyclopedia

of Cell Technology, John Wiley & Sons, New York, v. 2, pp. 853–872.

Walsh G (2004), Modern antibody based therapeutics, BioPharm. International,

December, 18–25.

Whalen, R.G. 1996. DNA vaccines for emerging infectious diseases: What if? Emerg.

Infect. Dis. 2: 168-175.

Witkowski JA (1979), Alexis Carrel and the mysticism of tissue culture. Med. Hist.

23:279–296.

12 Animal Cell Technology

Animal cells:

basic concepts

Patrı

cia Le

o, Adriana Lages Lima Galesi,

Cla

udio Alberto Torres Suazo, and A

ngela Maria Moraes

2.1 Introduction

The term cell culture refers to the cultivation of dispersed cells taken from

an original tissue, a primary culture, or a cell line. As mentioned in

Chapter 1, the practice of cultivating cells started at the beginning of the

20th century and was developed from a simple exploratory phase to an

expansion phase in the 1950s. For more than 50 years, the culture of cells

derived from primary tissue explants has predominated, justifying the

original name ‘‘tissue culture.’’ Currently, cell culture is in a specialization

phase. With the increase of the use of dispersed cells since the 1950s, the

term tissue culture was substituted by cell culture.

At the present time, cell culture techniques allow in vitro propagation of

various cell lines including those from insects, humans, mice, rats, and

other mammals. Basically, animal cell culture techniques are similar to

those employed for bacteria, fungi, and yeast, although there are some

characteristic differences. In general, animal cells are more delicate, vulner-

able to mechanical damage, present lower growth rates, and require more

complex culture media and special substrates (Augusto and Oliveira,

2001). Moreover, cell culture has to be performed under rigorous aseptic

conditions, since animal cells grow more slowly than most usual contami-

nants, such as bacteria and fungi.

The goal of this chapter is to discuss animal cell culture characteristics,

focusing on typical cell structural and morphological aspects, the establish-

ment, maintenance, and storage of cell lines, in vitro cell growth phases,

the effects of environmental conditions on cell cultivation, and culture of

anchorage-dependent cells, with emphasis on maintaining cell lines em-

ployed for the production of commercially attractive biomolecules. Cell

cycle phases (G0, G1, S, G2, and M), through which cells pass during

exponential growth, are discussed in detail in Chapter 7.

2.2 Typical structure of an animal cell

Various animal cell lines can be cultivated in vitro, such as cardiac cells,

ﬁbroblasts, smooth muscle cells, endocrine cells (such as pituitary, adrenal,

and pancreatic cells), epithelial cells (such as liver, mammary, lung, and

kidney cells), tumor cells (such as melanocytes), nervous system cells (such

as glial cells and neurons), as well as hybridomas. Although these cells

have different origins and functions, they all show a typical structure

represented in Figure 2.1.

Animal cells have two very distinct compartments, the cytoplasm and

the nucleus, with a constant ﬂow of distinct chemical compounds between

them. While the cytoplasm is enclosed in a plasma membrane, the nucleus

is enclosed by a nuclear envelope. The different organelles of the cell are

immersed in the cytoplasm. Each of these cellular components has speciﬁc

characteristics and functions that will be discussed below.

2.2.1 Plasma membrane

The cell membrane, also called the plasma membrane, surrounds the

cytoplasm and physically separates the intracellular components from the

extracellular environment. It has a thickness around 7.5–10 nm and

controls the ﬂux of molecules between the cytosol and the extracellular

environment. The plasma membrane is composed of a lipid bilayer, in

which the polar groups are exposed to the internal and external cell

aqueous medium, protecting the embedded hydrophobic tails. Various

proteins with distinct functions can be found partially or totally immersed

in this bilayer.

The lipid bilayer has two important properties. First, its interior hydro-

phobic environment acts as an impermeable barrier that prevents the

endosome

mitochondria

peroxisomes

plasma

membrane

free

polyribosomes

15 mµ

rough endoplasmic

reticulum with membrane-

bound polyribosomes

nucleus

Golgi complex

lysosome

cytosol

Figure 2.1

Main intracellular compartments of an animal cell. Cytosol, endoplasmic

reticulum, Golgi complex, nucleus, mitochondria, endosome, lysosome, and

peroxisome are distinct compartments that are isolated from the rest of the cell by

at least one selective membrane.

14 Animal Cell Technology

diffusion of hydrophilic solutes through the membrane. This imperme-

ability is selectively modulated by the presence of membrane proteins,

which mediate the transport of speciﬁc molecules through the plasma

membrane. The second bilayer property is its structural stability, main-

tained by hydrophobic and van der Waals interactions formed between

the lipid chains. Even if the external aqueous environment varies with

respect to ionic strength and pH, the lipid bilayer has the capacity to

maintain its characteristic architecture.

2.2.2 Cytoplasm

The eukaryotic cell cytoplasm consists of the cytosol, which is the matrix

that presents no visible structure even when observed by electron micro-

scopy. Organelles and inclusion bodies are immersed in the cytosol. The

organelles are responsible for intense metabolic activity and include the

mitochondria, Golgi complex, lysosome, and endoplasmic reticulum (Fig-

ure 2.1). Inclusion bodies are less frequent and consist of lipid, carbo-

hydrate, and pigment-storing structures.

The cytoplasm usually has a thin peripheral area in contact with cell

membrane, termed the ectoplasm. This area has few organelles and inclu-

sion bodies, which tend to be located in the central part of the cytoplasm,

named the endoplasm.

Although the basic architecture of all eukaryotic cells is formed by

membranes, organelles, and cytosol, each cell type exhibits a distinct

morphology deﬁned by cell shape and localization of organelles. The

structural basis of the characteristic morphology of each cell type is the

cytoskeleton, a dense network of three classes of ﬁlamentous proteins that

permeate the cytosol and support the cell membrane.

2.2.3 Endoplasmic reticulum

This is an interconnected network of ﬂattened or spherical vesicles and

tubules found in the cytoplasm of eukaryotic cells. These structures are

enveloped by a membrane that separates the endoplasmic reticulum

cavities or cisternae. The cisternae constitute a network of channels that go

through the cytoplasm and regulate the transport of various cell products,

generally to the exterior environment. In some cells the cisternae also serve

as a storage area. There are two types of endoplasmic reticulum: granular

(or rough) and smooth.

The rough endoplasmic reticulum contains ribosomes on the surfaces, a

cell structure that will be discussed in the next section. The system formed

by endoplasmic reticulum and ribosomes is associated with protein synth-

esis. The endoplasmic reticulum also participates in lipid biosynthesis. In

different types of eukaryotic cells, the endoplasmic reticulum has different

forms and functions. In muscle cells, in which Ca

2þ

stimulates contraction,

the endoplasmic reticulum participates in the relaxation process, reabsorb-

ing Ca

2þ

ions.

The smooth endoplasmic reticulum also consists of a network of tubules

that is considerably developed in certain types of special cells, such as

those that secrete steroid hormones.

Animal cells: basic concepts 15

2.2.4 Ribosome

Ribosomes are spherical cell structures enriched with ribonucleoprotein

and with a diameter of 15–20 nm. Each ribosome is formed by two

subunits of different sizes, only visible through high resolution electron

microscopy. Ribosomes connected to the rough endoplasmic reticulum

are involved in the biosynthesis of proteins that are temporarily stored or

transported to the cell’s outer environment. Proteins synthesized by

ribosomes connected to membranes accumulate in the cisternae before

passage to the plasma membrane for secretion.

2.2.5 Golgi complex

This organelle is also known as the Golgi or Golgi apparatus and is named

after the Italian cytologist Camilo Golgi, who was one of the ﬁrst to study

it.

The Golgi has distinct forms in different cell types. The most character-

istic arrangement is a stack of circular ﬂattened vesicles, with variable

sizes, each one held by a single membrane in which can be found smaller

spherical vesicles that bud off from the larger ones. In many cells, the

Golgi complex is situated near the cell nucleus but in other cells, it is

dispersed in the cytoplasm.

The Golgi complex receives products from the endoplasmic reticulum

and packs them in secretory vesicles, which are conducted and fused to the

external cell membrane. In this process, termed exocytosis, the vesicle

content is secreted into the outer environment.

2.2.6 Mitochondria

Mitochondria are spherical or, more frequently, elongated bodies, consist-

ing of two membrane units. The inner one is folded in the form of tubules

or shelves, named cristae.

Mitochondria are enzyme-rich structures that catalyze the oxidation of

organic nutrients by oxygen molecules, producing carbon dioxide and

water. Some of these enzymes are located in the matrix and some in the

internal membranes. Chemical energy is released during these oxidation

processes and is used to generate adenosine triphosphate (ATP), the most

important cell energy carrier molecule. ATP synthesized by mitochondria

diffuses throughout the cell and is utilized for many cellular functions.

The mitochondria also contain small quantities of DNA, as well as

RNA and ribosomes. Mitochondrial DNA encodes the synthesis of cer-

tain speciﬁc inner cell membrane proteins. Mitochondria can also divide

during cell replication.

2.2.7 Lysosome

Lysosomes are spherical organelles, enveloped by a membrane and con-

taining several hydrolytic enzymes that present maximal activity at acidic

pH. These enzymes are synthesized in the rough endoplasmic reticulum.

Lysosomes are reservoirs of different enzymes capable of degrading

16 Animal Cell Technology

macromolecules by hydrolysis such as proteins, polysaccharides, and

lipids. As these enzymes may be harmful to other cell components, they

remain segregated in the lysosomes. Proteins and other components to be

degraded are selectively transported to the lysosomes and are then hydro-

lyzed to their fundamental units, which are afterwards secreted into the

cytoplasm.

2.2.8 Peroxisome

Peroxisomes or microbodies are spherical organelles that are 0.3–1.5 m

in diameter. Each peroxisome is enveloped by a single external membrane,

and its interior is full of proteins, frequently in crystalline form. Peroxi-

somes are characterized by the presence of various oxidative enzymes,

which have variable functions dependent upon the origin of the peroxi-

some. These enzymes generate and utilize hydrogen peroxide (H

hence the name peroxisome. This compound is very toxic for cells and is

decomposed by the enzyme catalase to water and oxygen.

2.2.9 Nucleus

The nucleus of eukaryotic cells is a very complex structure, containing

various components. It is separated from the rest of the cell by two

membranes named the nuclear envelope. At regular intervals, the two

membranes of the nuclear envelope form pores with a diameter of around

90 nm. These pores regulate ﬂux of macromolecules to and from the

cytoplasm. Inside the nucleus is located the nucleolus, which acts to

produce ribonucleic acid (RNA), which is the ﬁrst step for ribosome

synthesis.

Chromatin occupies the remaining part of the nucleus and consists of

DNA, RNA, and specialized proteins. Between cell divisions, chromatin

is dispersed in the nucleus but, immediately before cell division, chromatin

is arranged in granular bodies named chromosomes. After replication,

daughter chromosomes are separated and distributed to daughter cells

during the mitosis process, after which the chromatin is dispersed again.

2.3 Cell culture

2.3.1 Establishing a cell line

Cells can be cultivated in vitro after a tissue is dissected and totally or

partially disaggregated by enzymic treatment. Cell, explants, or organ

cultures can be established depending on the degree of disaggregation of

the tissue, as shown in Figure 2.2.

Primary cultures are those obtained directly from organ or tissue

fragments, following enzymic or mechanical disaggregation. For enzymic

treatment, trypsin or collagenase may be used and may be mixed with

EDTA (ethylenediaminetetraacetic acid). Primary cultures can originate

from animal or human tissue. A culture is considered a primary culture up

to its ﬁrst subculture or passage; after that it is named a cell line. Figure 2.3

Animal cells: basic concepts 17

Cell

culture

Dissection

Adult

Egg

Enzymatic

digestion

Finely chopped

tissue

Further

dissection

Primary

explant

Organ

culture

Embryo

Figure 2.2

In vitro culture types of animal cells (adapted from Freshney, 2000, 2005).

Theoretical cell yield

(Log [cell number])

8121620

Culture time (weeks)

Primary

culture

Finite cell line

Continuous cell line

1 Subculture

2 Subculture

TRANSFORMATION

Subculture

interval

Serial

passages

SENESCENCE

AND DEATH

Figure 2.3

Typical evolution of a cell line (adapted from Freshney, 2000, 2005).

18 Animal Cell Technology

shows a schematic diagram of the sequence of events associated with

subculture of a normal primary cell culture.

Primary cultures are initially heterogeneous, but ﬁbroblasts may be-

come predominant after some growth. Obtaining primary cultures is

laborious and cells can be maintained in vitro for only a limited period.

During this period, cells generally maintain the differentiated character-

istics of the original tissue from which they were harvested.

During primary culture, cells may be subjected to considerable stress.

For instance, the enzymatic dissociation of organ fragments or adherent

cells breaks cell–cell or cell–surface interactions. Dissociated cells gener-

ally change their shape, becoming rounded and losing their phenotypic

polarity, modifying protein distribution in cell membrane.

Certainly not all cells survive cell manipulation but those that survive

should be able to correct any injuries and adapt to environmental changes.

Culture adaptation is time-consuming and is inﬂuenced by culture condi-

tions. Cells condition their environment through the release of substances

into culture medium, such as growth factors, which promote cell adher-

ence and proliferation. Some cell lines can adapt, proliferate, and differ-

entiate faster if culture medium is previously conditioned by cells in active

growth. Environmental conditioning can also be achieved by adding cells

to medium already inoculated with a non-mitotic viable cell population,

named feeder cells. These cells can be prepared from 3T3 ﬁbroblastic cells

exposed to gamma radiation or treated with mitomicin C (Rheinwald,

1989).

After successive subcultures of a very heterogeneous primary culture,

containing many cell types of the original tissue, a more homogeneous cell

line with a higher growth rate may arise. A cell line can be serially

propagated in culture, usually for only a limited number of cell divisions.

Finite cell lines are generally diploid and maintain some degree of

differentiation. Nevertheless, these cell lines die after a limited number of

generations, the Hayﬂick limit, which is usually about 30–50 division

cycles depending on the origin of the cells (Hayﬂick and Moorhead, 1961).

The immortalization of a cell line can be accomplished as a spontaneous

process, by an oncogene or virus or by chemical treatment. This can lead

to a continuous cell line that can be propagated for an undetermined

period. If such changes occur with an affect on cell cycle control, the cell

line can be designated a transformed cell line.

According to Freshney (2000), the main characteristics of transformed

cell lines are: (i) altered cell morphology (smaller, less adherent, or more

rounded cells, with a higher nucleus to cytoplasm ratio); (ii) higher growth

rate (duplication times decrease from 36–48 hours to 12–36 hours); (iii)

less dependency on blood serum or selected growth factors; (iv) increase

in cloning efﬁciency; (v) increase in heteroploidy (chromosomal variation

between cells) and in aneuploidy (divergence from the original diploid

number); (vi) increase in tumorigenicity. Some of these characteristics are

associated with malignant transformations. The main advantage of trans-

formed cells for cell culture is the almost unlimited cell supply. However,

a disadvantage is that the cells generally maintain few characteristics of

their original tissue.

Animal cells: basic concepts 19

2.3.2 Cell line maintenance

When a primary culture or a cell line is initiated, a periodic change of

culture medium is necessary, followed by subculture if the cells are

proliferating. Culture medium changes should be performed even in

cultures showing no cell proliferation, since cells can metabolize and

deplete nutrients from the medium. Also, some nutrients may degrade

spontaneously. Intervals between medium changes and subcultures may

vary depending on the cell line, growth rate, and metabolism. An increase

in cell density, pH decrease, nutrient depletion in the medium, or altera-

tion in cell morphology indicate the need for culture medium replacement.

Animal cells may be anchorage-dependent. Cells that depend on a solid

substratum for growth are named adherent cells. These cells normally

proliferate in monolayers and show contact inhibition, with the maximum

cell yield generally limited by the available surface of the culture vessel.

The yield of cells in suspension is not dependent on a solid substratum.

Table 2.1 shows some adherent and suspension cell lines that are

commonly cultured. Cell shape usually reﬂects their origin. Blood cells

(such as those derived from lymphomas) generally grow in suspension,

while cells derived from solid tissues (such as kidney and liver) are

adherent cells. Typical morphologies of different cell types are shown in

Figure 2.4 (see color section).

Since adherent cells proliferate only after cell surface adhesion, an

understanding of the steps of this process is very important. The ﬁrst step

consists of the adsorption of adhesion factors to the substratum, such as

Table 2.1 Adherent and suspension cell lines commonly employed in animal

cell culture

Name Cell type and tissue origin Morphology

Adherent cells

MRC-5 Human lung Fibroblast

HeLa Human cervix Epithelial

Vero African green monkey kidney Epithelial

NIH 3T3 Mouse embryo Fibroblast

L929 Mouse connective tissue Fibroblast

CHO Chinese hamster ovary Fibroblast

BHK-21 Syrian hamster kidney Fibroblast

HEK-293 Human kidney Epithelial

HEPG2 Human liver Epithelial

BAE-1 Bovine aorta Endothelial

Suspension cells

NS0 Mouse myeloma Lymphoblastoid-like

U937 Human hystiocytic lymphoma Lymphoblastoid

Namalwa Human lymphoma Lymphoblastoid

HL60 Human leukemia Lymphoblastoid-like

WEHI 231 Mouse B-cell lymphoma Lymphoblastoid

YAC 1 Mouse lymphoma Lymphoblastoid

U 266B1 Human myeloma Lymphoblastoid

SH-SY5Y Human neuroblastoma Neuroblast

Adapted from ECACC Handbook, 2005.

20 Animal Cell Technology

vitronectin and/or ﬁbronectin glycoproteins and often associated to Ca

2þ

ions. These factors can be derived from serum or can be produced by the

cells themselves. The second step consists of contact of the cell with the

surface, while in the third step, cells attach to the covered surface,

producing multivalent heparin sulfate proteoglycans, which bind to cell

membrane glycoproteins. In the fourth step, cell spreading over the solid

surface occurs.

As vertebrate cells have negative charges non-uniformly distributed over

their external membrane surface, solid substrata with a hydrophilic surface

are required, with an adequate distribution of the surface charge.

For the subculture of adherent cells, removal of culture medium and the

detachment of cells from the monolayer are necessary. This detachment is

usually performed with trypsin, but other proteases, such as pronase,

dispase, and collagenase, can be employed. In general, a chelating agent,

such as EDTA, is also added to capture the Ca

2þ

ions involved in the cell

adhesion process. Some cell lines bind weakly to surfaces and, in small-

scale cultures, can be removed mechanically by gently tapping or hitting

the culture ﬂask by hand.

For cells growing continuously in suspension, the subculture process

can be performed similarly to the method used for microbial cultures.

Trypsin treatment is not required and subculture is faster and less

traumatic for the cells. Total medium exchange is not generally performed

for these cultures since it would require a centrifugation step. Culture

maintenance can be performed by dilution with fresh medium after

adequate cell growth.

Some cell lines can proliferate as semi-adherent cells, and the population

is a combination of suspended and adherent cells. To maintain the hetero-

geneous nature of such cultures, both suspended and adherent cells should

be subcultured.

Suspension cultures present some advantages in comparison with adher-

ent cell cultures. Large cell quantities can be attained without increasing

the superﬁcial area of the substratum. Also, a steady state can be reached if

nutrients are continuously supplied and cell concentration is kept con-

stant. This can rarely be achieved with a monolayer culture. Cells in

different modes (adherent or suspension) behave differently with regard to

proliferation, enzymatic activity, glycolysis, respiration, specialized pro-

duct synthesis, and many other properties.

2.4 Cell growth phases

Normal cells in culture show a sigmoid pattern of proliferative activity

that reﬂects culture adaptation, environmental conditioning, nutrient

availability and, for adherent cells, available free adhesion surface.

Cell growth phases comprise lag phase, exponential or log growth

phase, stationary or plateau phase, and senescence or death phase, as

shown in Figure 2.5. Cell growth can be mathematically represented by

the following general equation:

¼ :X (1)

Animal cells: basic concepts 21