Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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BACKGROUND OF TH~ INVENTION
1 Field of the Invention
This invention is in the field of biology and more
particularly in the field of cell biology.
2. Descripti~n of the Prior Art
The ability to grow mammalian cells is important
at both the laboratory and industrial levels. At the labora- -
tory level, the limiting factor for cellular or viral re-
search at the sub-cellular level is often the amount of raw
material available to be studied. At the industrial level,
there is much effort being devoted to the development of
pharmaceuticals based on mammalian cell products. These are
primarily vaccines for human or animal viruses, but also
include human growth hormone and other body hormones and bio-
chemicals for medical applications.
; Some mammalian cell types have been adapted for
growth in suspension cultures. Examples of such cell types
include HeLa ~human), BHR (baby hamster kidney) and L cells
(mouse). Such cells, in general, have non-normal genetic
complements, i.e., too many or too few chromosomes or ab-
normal chr~mosomes. Often, these cells will produce a tumor
upon injection into an animal of the appropriate species.
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Other mammalian cell types have not been adapted for
growth in suspension culture to date, and will grow only if
they can become attached to an appropriate surface. Such cell
types are generally termed "anchorage-dependent" and include
3T3 mouse fibroblasts, mouse bone marrow epithelial cells;
Murine leukemia virus-producing strains of mouse fibroblasts,
primary and secondary chick fibroblasts; WI-38 human fibroblast
cells; and, normal human embryo lung fibroblast cells (HEL299,
ATCC #CCL137). Some anchorage-dependent cells have been grown
which are tumor causing but others were grown and found to be
non-tumor causing. Also, some anchorage-dependent cells, such
as WI-38 and HEL299, can be grown which are genetically normal.
Whereas considerable progress has been made in large
scale mammalian cell propagation using cell lines capable of
growth in suspension culture, progress has been very limited
for large scale propagation of anchorage-dependent mammalian
cells. Previous operational techniques employed for large
scale propagation of anchorage-dependent cells were based on
linear expansion of small scale processes. ~Cell~culture plànts
utilized a large number of low yield batch reactors, in the forms
of dishes, prescription bottles, roller tubes and-~roller bottles.
Each of these was a discrete unit or isolated batch-reactor requi
ing individual environmental controls. These controls, however,
were of the most primftive type due to economic considerations.
Variation in nutrients was corrected by a medium change, an
operation requiring two steps, i.e., medium removal and meaium
addition. Since it was not uncommon for a moderately sized
facility to operate hundreds of these batch reactors at a time,
even a single change of medium required hundreds of operations,
all of which had to be performed accurately, and under exact-
ing fiterile conditionfi. Any multiple step operation, such as
cell transfer or harvest, compounded the problem accordingly.
Thus, co~ts of equipment, space and manpower were great for this
type of facility.
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There are alternative methods to linear scale-up
from small batch cultures which have been proposed. Among
such alternatives which have been reported in the literature
are plastic bags, stacked plates, spiral films, glass bead
propagators, artificial capillaries, and microcarriers.
Among these, microcarrier systems offer certain outstanding
and unique advantages. For example, great increases in the
attainable ratio of growth surface to vessel volume (S/V) can
be obtained using microcarriers over both traditional and newly
developed alternative techniques. The increase in S~V
attainable allows the construction of a single-unit homogeneous
or quasi-homogeneous batch or semi-batch propagator for high
volumetric productivity. Thus, a single stirred tank vessel
with simple feedback control for pH and PO2 presents a homo-
geneous environment for a large number of cells thereby elimina-
ting the necessity for expensive and space consuming, controlled
environment incubators. Also, the total number of operations
required per unit of cells produced is drastically reduced.
In summary, microcarriers seem to offer economies of capital, -
space and manpower in the production of anchorage-dependent
cells, relative to current production methods.
Microcarriers also offer the advantage of environ-
mental continuity since the cells are grown in one controlled
environment. Thus, microcarriers provide the potential for
growing anchorage-dependent mammalian cells under one set of
environmental conditions which can be regulated to provide
constant, optimal cell growth.
One of the more promising microcarrier systems to
date has been reported by van Wezel and involves the use of
diethylaminoethyl (DEAE)-substituted dextran beads in a
stirred tank. A. L. van Wezel, "Growth of Cell Strains and
Primary Cells on Microcarriers in Homogeneous Culture", Nature
216:64 (1967); D. van Hemert, D. G. Kilburn and A. L. van Wezel,
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"Homogeneous Cultivation of Animal Cells for the Production
of Virus and Virus Products", Biotechnol. Bioeng. 11:875
(1969); and A. L. van Wezel, "Microcarrier Cultures of
Animal Cells", Tissue Culture, Methods and Applications,
P. F. Kruse and M. K. Patterson, eds., Académic Press,
New York, p. 372 (1973). These beads are commerically
produced by Pharmacia Fine Chemicals, Inc., Piscataway,
1~ ~r~o/en7ar/~
1~ New Jersey, under the tradonamo DEAE-Sephadex A50, an ion
exchange system. Chemically, these beads are formed from a
crosslinked dextran matrix having diethylaminoethyl groups
covalently bound to the dextran chains. As commercially
available, DEAE-Sephadex A50 beads-are believed-to have a
particle size of 40-120u and a positive charge capacity of
about 5.4 meq per gram of dry, crosslinked dextran (ignores
weight of attached DEAE moieties).-- Other anion exchange
resins, such as DEAE-Sephadex A25, QAE-Sephadex A50 and
QAE-Sephadex A25 were also stated by van Wezel to support
cell growth.
The system proposed by van Wezel combines multiple
surfaces with movable surfaces and has the potential for
innovative cellular manipulations and offers advantages in
scale-up and environmental controls~ Despite this potential, `
these suggested techniques have not been significantly ex-
ploited because researchers have encountered difficulties
in cell production due to certain deleterious effects caused
by the beads. Among these are initial cell death among a
high percentage of the cell inoculum and inadequate cell
growth even for those cells which attach. The reasons for these
deleterious effects are not thoroughly understood, although
it has been proposed that they may be due to bead toxicity or
nutrient adsorption. See van Wezel, A. L. ~1967), Nature 216:
64-65; van Wezel, A. L. (1973), Tissue Culture, Methods and
Applications. Kruse, P. R. and Patterson, M. R. ~eds.),
. . .
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pp. 372-377, Academic Press, New York; van Hemert, P., Rilburn,
D. G., and van Wezel, A. L. (1969), Biotechnol. Bioeng. 11:
875-885; Horng, C. and McLimans, W. (1975), Biotechnol.
Bioeng. 17: 713-732.
It could be that the deleterious effects of these
commercially available ion exchange resins are due to their
method of manufacture. Certain of these production methods
are descri~ed for polyhydroxy materials in patents such as:
U. S. 3,277,025; 3,275,576; 3,042,667 and 3,208,994 all to
Flodin et al. Whatever the reason, however, the presently
commercially available materials are simply not sufficient
for good cell growth of a wide variety of cell types.
One solution to overcoming some of the deleterious
effects encountered in attempts to use such commercially
available microcarriers for cell growth is described in
U.S. Patent No. 4,036,693, issued on July 19, 1977 to
Levine et al. Therein, a method for treating these com~er-
cially available ion exchange resins with macromolecular
polyanions, such as carboxymethylcellulose, is proposed.
While this method has proven successful, it would clearly
be more advantageous if the beads could be manufactuxed
initially to have properties designed for outstanding
growth of anchorage-dependent cells.
SUMMARY OF IHE INVENTION
2$ It has now been discovered that the charge
capaci~ty of microcarriers has to be adjusted and/or con-
trolled within a certain range to result in good growth of
a wide variety of anchorage-dependent cell types at
reasonable microcarrier concentrations. Based upon this
discovery, microcarrier beads have been produced with con-
trolled charge capacities and such beads have been used to
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obtain good growth of a variety of anchorage-dependent cells.
Cells grown using such microcarrier systems can be harvested
or used in the production of animal or plant viruses, vaccines
hormones, interferon or other cell growth by-products.
The invention relates to a method of growing anchorage-
dependent cells in microcarrier culture, the improvement of em-
ploying microcarriers having an amount of positively charged
chemical moieties thereon adjusted to provide an exchange capac-
ity which allows good growth of said cells, said exchange capac-
ity being within the range of between about O.l and about 4.5meq/gram of dry, untreated microcarriers.
The invention also relates to cell culture micro-
carriers having a degree of substitution thereon with positively
charged chemical moieties sufficient to provide a charge capac-
ity at which good cell growth will occur, and which is from
about 0.1 to about 4.5 meq/gram of dry, untreated microcarriers.
One example of the improved microcarriers is those
produced using polymers with pendant hydroxy groups, such as
crosslinked dextran beads. These beads can be treated with an
aqueous solution of a tertiary or quaternary amine, such as
diethylaminoethylchloride:chloride, and an alkaline material,
such as sodium hydroxide. The specific charge capacity of the
beads is controlled by varying the absolute amounts of the dex-
tran, tertiary amine salt and alkaline material, the ratio of -
these materials, and/ox the time and temperature of treatment.
Microcarriers produced according to this invention
can be used in cultures without the high initial cell loss here-
tofore experienced with commercially available microcarriers.
Additionally, attached cells spread and grow to confluence on
the bead~ reaching extremely high cell concentrations in the
~uspending medium. The concentration of microcarriers in sus-
pen~ion is not limited to very low levels as was customary with
A ~ 7 _
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the prior art materials, and cell growth appears only to be
limited by factors which do not appear to be associated with the
microcarriers. Because of this, great increases in the volu-
metric productivity of cell cultures can be obtained. In short,
the potential offered from the use of microcarriers in the growth
of cells, and particularly anchorage-dependent cells, can now
be realized.
~ .... . .
A - 7a -
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- BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a plot graphically illustrating the
growth characteristics of normal diploid human embryo lung
fibroblast cells (HEL299) at a microcarrier concentration
of 2 grams dry, crosslinked dextran/liter for both commer-
cially available DEAE-treated dextran microcarriers and
DEAE-treated microcarriers produced according to this
invention;
FIG. 2 graphically illustrates the growth charac-
teristics of both normal diploid human embryo lung fibro-
blast cells (HEL299) and secondary chicken embryo fibro-
blasts at a microcarrier concentration of 5 grams dry,
crosslinked dextran/liter using improved DEAE-treated .:-
microcarriers of this invention.
DESCRIPTION OF PREFERRED EMBODIMENTS
As used herein, the terms "microcarriers~, "-cell- -
culture microcarriers" and "cell-growth microcarriers" .
mean small, discrete particles suitable for cell attachment
-- -and g~rowth-. -Often, alt~oug~-~ot always, microcarr-iers are -
porous beads which are formed from polymers. Usually,
cells attach to and grow on the outer surfaces of such beads.
As previously described, it has now been dis- : -
covered that the amount of charge capacity on cell culture
microcarriers must be adjusted and/or controlled to be
.~ :~ within a certain range for adequate cell growth`iat r ` , .
~reasonable microcarrier concentrations. Suitable oper-
ating and preferred ranges will vary with such factors as
the spec~f-ic cel~ls to-~e-grown, the nature of the micro-
carriers, the concentration of microcarriers, and other
culture parameters including medium composition. In all
cases-~-however~- the-amount of charge capacity which has - -- - -
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,, . . ~. :
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been found to be suitable is significantly below the
amounts believed to be present on commercially available
anion exchange resins previously suggested for microcarrier
cell cultures. For example, it is believed that the DEAE-
Sephadex A50 beads, suggested by van Wezel, have a charge
capacity of about 5.4 meq/gram of dry, untreated (without
DEAE), crosslinked dextran. In contradistinction to this
relatively high charge capacity, microcarriers have been
produced and found suitable for good cell growth according
to this invention which have between about 0.1 and about
4.5 meq/gram of dry, untreated microcarriers. Below about
0.1 meq/gram, it is believed that cells would have
difficulty attaching to the microcarriers. Above about
4.5 meq/gram, losses of initial cell inoculum ta~e place,
and even the surviving cells do not grow well,
particularly at relatively high microcarrier concentrations.
For the growth of normal diploid human fibro-
blasts on crosslinked dextran microcarriers, it has been
found that a preferred range of charge capacity supplied by
DEAE groups is from about 1.0 to about 2.8 meq/g~ of dry,
untreated crosslinked dextran. While the preferred range
may vary with different cell types or culture conditions, ~ -
it is believed that the preferred ranges for any given set
of conditions will be within the 0.1-4.5 meq/~m range.
The preferred and optimum conditions can be determined by
a person skilled in the art for any set of conditions by
routine experimentation.
It will be recognized, of course, that there are
certain deficiencies in attempting to define the charge
capacity of microcarriers strictly on a unit weight basis.
For example, two beads identical in every way except that
they are formed from material~ having different densities
: .: . .. : : .. .. . : : - . . . , . .: ~ . . . .. .. .
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with the same charge distribution thereon would yield
different values for their charge capacity per unit weight.
Similarly, two beads having identical charge capacities
per unit weight might have quite different charge distribu-
tions thereon.
An-alternative definition can be made by specify-
ing the range of suitable charges in terms of charge
capacity per unit weight of microcarriers in their final
functional form. This basis would take into account such
factors as the weight of attached DEAE or other positively
charged groups, as well as hydration of the beads, etc.,
whereas the prior-definition is-based-on-dry-, crosslinked -
dextran and does not take such factors into account. In
an aqueous cell culture medium, the density of micro-
carriers should be close to 1.0 gram/cc so that the micro-
carriers can be readily dispersed throughout the culture.
Based upon this, it has been determined that the range of
suitable charge capacities for microcarriers of this
invention defined in this way is from about 0.012 to
about 0.25 meq/gram.
The ranges of suitable çharge capacities previ- x
ously specified on a weight basis are valid assuming the
microcarriers have a substantially uniform charge distri-
bution throughout their bulk. If the charge distribution
is uneven, it might be possible to have suitable micro-
carriers having charge capacities outside of those ranges.
~he important criterion is, of course, that the charge
capacity be adjusted to and/or controlled at a value suffi-
cient to allow good cell growth on the microcarriers.
Since it may be the charge pattern on the outer
surface which is important, it i8 also desirable to be
able to define the suitable charge capacity range in
.
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terms of the likely surface pattern. This can be done by
assuming that the active portion of the microcarriers
represents only the outer surface of the bead to a depth
of about 20 angstroms. If it is also assumed that the
charged groups in the previously mentioned cases are
evenly distributed throughout the beads, the previous
ranges can be converted to a charge capacity in this outex
shell. Using this approach, the range of charge capacity
found suitable is from about 0.012 meq/cm3 to about 0.25
meq/cm3. This approach ta~es changes in microcarrier
volume due to different charge densities into account.
Microcarriers having the re~uired charge capacity
can be prepared by treating microcarriers formed from polymers
containing pendant hydroxyl groups with an aqueous solution
of an alkaline material and a tertiary or quaternary
amine. The beads can be initially swollen in an aqueous
medium without the other ingredients, or can be simply con-
tacted with an aqueous medium containing the required base and
amine. This method of using alkaline materials to catalyze
the attachment of positively charged amino groups to hydroxyl-
containing polymers is described in Hartmann, U. S. Patent
1,777,970.
Examples of suitable hydroxyl-containing polymers
include polysaccharides such as dextran, dextrin, starch, cellu-
lose, polyglucose and substituted derivatives of~these.
Certain synthetic polymers such as polyvinyl alcohol and
hydroxy-substituted acrylates or methacrylates, such as hydroxy-
ethyl methacrylate, are also suitable. Dextran, and especially
crosslinked dextran in the form of small spheres or beads, is
particularly preferred because it is commercially available,
relatively inexpensive, and produces microcarriers which support
excellent cell growth.
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Any material which is alkaline can be used for the
reaction. The alkali metal hydroxides, such as sodium or
potassium hydroxide, are, however, the preferred alkaline sub-
stances.
Either tertiary or quaternary amines are suitable
sources of positively charged groups which can be appended
onto the hydroxy-containing polymers. Particularly preferred
materials are chloro- or bromo-substituted tertiary amines or
salts thereof, such as diethylaminoethylchloride, diethylamino-
ethylbromide, dimethylaminoethylchloride, dimethylaminoethyl-
bromide, diethylaminomethylchloride, diethylaminomethylbromide,
di-(hydroxyethyl)-aminoethylchloride, di-(hydroxyethyl)~aminoethyl-
bromide, di-(hydroxyethyl)-aminomethylchloride, di-(hydroxy-
ethyl)-aminomethylbromide, f-morfolinoethylethylchloride~
~-morfolinoethylbromide, ~-morfolinomethylchloride, ~-mor-
- folinomethylbromide and salts thereof, for example, the
hydrochlorides.
.
A wide range of reaction temperatures and times
may be used. It is preferred to carry out the reactions at
temperatures of about between 18C and 65C. However, other
temperatures can be used. The reaction kine~ics depend to a
large extent, of course,- upon the reaction temperature and the
concentration of reactants. Both the time and temperature do
affect the final exchange capacity achieved.
The reason that-the charge capacity of the micro-
carriers is so critical in cell growth is not thoroughly
understood. While not wishing to be bound by this theory, it
is possible that the charge capacity at the surface causes certain
local discontinuities of medium composition which are the major
- controlling influence in microcarrier culture cell growth.
Nevertheless, this is not meant to rule out other possibilities.
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There may be certain bead~, of cour~e, that will not
be quitable for good cell growth even though they have a charge
capacity within one of the range~ specified. mi~ may be due
to 3ide chain~ on the moiety Rupplying the charge capacity
which are toxic or otherwise deleteriou~ for cell growth, the
presence of ad~orbed or ab~orbed deleterious composition~ or
compound~, or it may be due to the porosity of the bead or due
to other reason~. If ~uch bead3 are not suitable for cell
growth except for the amount of charge capacity, the beads are
not con~idered to be "cell-growth microcarriers."
m e invention is further illustrated by the following
examples.
EXAMPLE 1
_ EPARATION OF DMPROVED MICROCARRIERS
Improved microcarrier~ can be produced as follows.
Dry, uncharged, crosslinked dextran bead9 are sieved to obtain
those of approximately 75 um in diameter. One gram of this
fraction is added to 10 ml of distilled water and the beads are
allowed to swell. An adequate commercial source of dry, cros~-
linked dextran known under the trade mark Sephadex G-50 from
Pharmacia Fine Chemical~, Piscataway, ~ew Jersey.
An aquQous solution containing 0.01 moles of
diethylaminoethylchloride:chloride, tw~ce recrystallized from ~ -
methylene chloride, and 0.015 moles of sodium hydroxide is
formed in a 10 ml volume. This aqueous ~olution is then added
to the swollen dextran bead ~uspen~ion, which is then agitated
vig~rously in a shaking water bath for one hour at 60C. After
one hour, the beads are ~eparated from the reaction mixture
by filtration on Whatman ~trade mark) filter paper No. 595 and
wa~hed with 500 ml of distilled water.
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Beads made by this procedure contain approximately
2.0 meq of charge capacity per gram of dry, untreated cross-
linked dextran. ThiA charge capacity can be characterized by
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measuring the anion exchange capability of the beads as
follows. The bead preparations are washed thoroughly with
0.1 normal HCl to saturate all exchange sites with Cl ions.
They are then rinsed with 10-4 normal HCl to remove unbound
chloride ions. Subsequently, the beads are washed with a
10% Iw/w) sodium sulfate solution to countersaturate the
exchange sites with SO4=. The effluent of the sodium
sulfate wash is collected and contains liberated chloride
ions. This solut~on is titrated with 1 M silver nitrate
using dilute potassium chromate as an indicator.
After titration, the beads are washed thoroughly
with distilled water, rinsed with the phosphate-buffered
saline solution (PBS), suspended in PBS and autoclaved.
This procedure yields hydrated beads of approximately
120-200 pm in diameter, which carry about 2.0 meq of charge
capacity per gram of dry, untreated, crosslinked dextran.
EXAMPLE 2.
GROWTH OF ANCHORAGE-DEPENDENT CELLS
WITH MICROCARRIERS OF THIS INVENTION
CONTRASTED TO COMMERCIALLY AVAILABLE ION EXCHANGE RESIN
All cells were grown in Dulbecco's Modified
Eagle's Medium. For growth of normal diploid fibroblasts,
the medium was supplemented to 10% with fetal calf serum.
For growth of primary and secondary chicken fibroblasts,
the medium was supplemented with 1% chicken serum, 1% calf
serum, and 2% tryptose phosphate broth (~ifco Laboratories,
Detroit, M~). Stocks were passaged on 100-mm plastic
dishes ~Falcon Plastics, Inc., Oxnard, CA).
Primary chicken embryo fibroblasts were prepared
by mincing and ~equentially trypsinizing 10-day embryos.
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Secondary chicken embryo fibroblasts were prepared on the
first day of primary confluence by trypsinization. For cells
grown in plastic dishes, doubling time was about 20 hours.
Diploid human fibroblasts derived from embryonic
lung (HEL299, ATCC #CCL 137) were obtained from the American
Type Culture Collection, Rockville, MD. These cells had a
doubling time of 19 hours in plastic dishes.
Microcarrier cultures were initiated simply by combin-
ing cells and beads in stirred culture. 100-ml culture volumes
in 250-ml glass spinner bottles (6.5 cm in diameter) equipped
with a 4.5-cm magnetically driven TeflOn~ coate:d stir bar
(Wilbur Scientific, Inc.,-Boston7-MA~ were-used.- Stirring --
speed was approximately 90 rpm. Cultures were sampled directly,
and samples were examined microscopically and photographed.
Cells were enumerated by counting nucleii using the modifi-
cation of the method of Sanford et al. (Sanford, K. K., Earle,
W. R., Evans, V. J., Waltz, H. K., and Shannon, J. E. (1951) -J. Natl Cancer Inst. 11: 773.) as described by van Wezel
(van Wezel, A. L. (1973). Tissue Culture, Methods and Appli- -
cations. Kruse, P. F. and Patterson, M. R. (eds.), pp. 372-
377, Academic Press, New York).
- - Beads with-attached cells were separated from the
culture medium by permitting the beads to settle at 1 g for
a few minutes and then aspirating the supernatant. This
proaedure greatly facilitated the replacement of medium as
well as facilitating the separation of cells from micro-
carriers after trypsinization.
- Commerclal D~AE Sephadex A-50 was used as micro-
carrier for the diploid human fibroblasts and compared with
carrier~ 8ynthesiZed and titrated as described in Example 1.
- Por-both bead types~ carrier concentration was 2 gram~ of
dry, untreated, crosslinked dextran per liter. The charge
capacity of the DEAE Sephadex A-50 was 5.4 meq/g of dry,
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. . : ,: ~ . - . ,
-- 1~63050
crosslinked dextran, while that of the newly synthesized
beads was 2.0 meq/g. The results are illustrated in FIG. 1.
For this cell type, loss of original inoculum on
A-50 microcarriers was marked, while the fibroblasts attach,
proliferate, and reach confluence on the microcarriers of
this invention in six days. This behavior agrees well with
the reported behavior of this cell type on standard plates.
As FIG. 1 shows, the final cell density achieved with the
new microcarriers at 2 grams dry, crosslinked dextran/liter
was 1.2 x 106 cells/ml.
Cultures containing the new carriers demonstrated
neither initial cell loss nor any inhibition in~reaching
confluence. More importantly, the cultures grew normally
at higher microcarrier concentrations. In FIG. 2, for ~
example, human fibroblasts and secondary chicken embryo ~ -
fibroblasts are shown to reach saturation concentrations
near 4 x 106 cells/ml when 5 grams of dry, crosslinked
dextran per liter were used with the new carriers having a
charge capacity of 2.0 meq/g dextran. As can be seen, even -
at this relatively high microcarrier concentration, there
was no significant loss of inoculum.
Secondary chick embryo fibroblasts were also
grown at a microcarrier concentration of 10 grams/litex.
With the conditions described above, a saturation concen-
tration of 6 x 106 cells/ml was achieved; with addition to
~ the medium of an additional 1% fetal calf serum, a~ ;
; saturation concentration of 8 x 106 cells/ml was achieved.
There was no significant loss of cell inoculum.
Primary chick embryo fibroblasts were grown at a
microcarrier concentration of 5 and lO grams/liter and the
growth characteristics were similar to those of~the
~econdary chick fibroblasts, although slight inoculum
losse~ were noted ana somewhat longer lag times were
encountered.
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Attempts were also made to grow secondary chick
embryo fibroblasts under conditions similar to those used
above except that DEAE-Sephadex A-50 microcarriers at con-
centrations of 1 and 5 grams/liter were used. No cell
growth was detected and significant inoculum loss occurred.
EXAMPLE 3.
PREPARATION OF MICROCARRIERS
WITH VARYING AMOUNTS OF REACTANTS
Batches of microcarriers were prepared by dissolv-
ing diethylaminoethylchloride:chloride and sodium hydroxide
in 20 ml of distilled water. The-solution was then-poured -
over dry Sephadex G-50 beads after which the beads were
placed on a reciprocating shaker-water table maintained at
60C. One set of bead batches was treated with a solution
containing 0.01 moles of the amine and 0.015 moles of sodium
hydroxide, whereas another set of batches was treated with a
solution containing 0.03 moles of the amine and 0.045 moles
of sodium hydroxide. The reaction time was varied to produce
different meq/g within each batch.
Diploid human fibroblasts ~HEL299) were grown in
suspension cultures at a microcarrier concentration of 5.0
grams dry, untreated crosslinked dextran per liter following
the procedures of Example 2 using microcarriers having varying
me~/gram selected from each batch. Su~bsequently, productivity -
(106 cells grown/liter hour) was calculated and plotted versus
meq/gram for each batch of beads produced as above. Curves
plotted using data obtained for both sets were similar in shape,
having a general bell shape, but the curve from the batches
treated with the higher concentration of reactant~ had a some-
what 8harper rise and fall. ~arriers yieldin~ excellent cell
growth were produced from each batch.
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EXAMPLE 4.
PREPARATION OF MICROCARRIERS
AT VARYING A~INE/ALKALI RATIOS
This example illustrates further changes in the
charge capacity which can be obtained by varying DEAE chloride:
chloride/NaOH ratios. In this example, the procedures of
Example 3 were followed except that a wide range of concentra-
tions of sodium hydroxide was used while maintaining the con-
centration of the diethylaminoethylchloride:chloride at 0.01
moles per 20 ml. The concentrations used for the sodium hy-
droxide were 0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.02,
0.03, 0.05, 0.75, 0.10 moles per 20 ml.-
A plot was made of meq/gram after 1.25 hours at
60C. versus concentration of sodium hydroxi~e. It was ob-
served from the plot that concentrations of sodium hydroxide
below about 0.01 produced no detectable charge capacity.
Charge capacity rose quickly, however, with increases in
concentration and reached a maximum of around 2.3 meq/gram dry,
crosslinked dextran at a concentration of about 0.014 moles sodium
hydroxide. Charge capacity then declined in an almost linear
relationship to a value of about 1.1 meq/gram at a sodium
hydroxide concentration of about 0.10 moles. Thus, a change
in reaction kinetics takes place when the ratio of DEAE Chloride: -
chloride to sodium hydroxide is varied at a constant concentra-
tion of DEAE chloride:chloride and crosslinked dextran.
EXAMPLE 5.
HUMAN INTERFERON PRODUCTION IN CELLS
GROWN ON IMPROUED MICROCARRIERS
~ The ability of microcarrier grown cells to produce
;~ 30 human interferon is described herein. Cells used for the pro-
.
duction of human interferon were normal diploid human foreskin
fibrobla~ts, FS-4. The~e fibroblasts were grown in micro-
carrier culture~ using procedures as in Example 2. Microcarriers
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prepared and titrated according to Example 1 were used at a
concentration of 5 grams of dry, crosslinked dextran/liter.
The medium used for culture growth was DMEM supplemented
with 10% fetal calf serum.
In 8 to 10 days, cultures ceased growing. At this
point, growth medium was removed. Cultures were washed 1-4
times with 100 ml of serum-free DMEM. The cells were then
ready for interferon induction. This was accomplished by
adding to the cultures 50 ml of serum-free DMEM medium con-
taining 50~ug/ml cyclohexamide, and varying amounts of poly I
poly C inducer. After 4 hours, Actinomycin D was added to
the cultures to a final concentration of 1 ~g/ml.
Five hours after the onset of induction, inducing
medium was decanted and cultures were washed 3-4 times
with 100 ml of warm serum-free DMEM. Cultures were replenished
with 50 ml of DMEM containing 0.5% human plasma protein. ~ -
Cultures were incubated under standard conditions for an addi-
tional 18 hours. At this time, cultures were decanted, and -
the decanted medium was assayed for interferon activity. -
Interferon activity was assayèd by determining the 50% level of
cell protection for samples and standard solutions, for FS-4
fibroblasts challenged with Vesicular Stomatitis Virus (VSV),
Indiana strain. The results of interferon production runs
are presented in tabular form below.
25 InducerCell Concentration
ConcentrationDuring ProductionInterferon
~pg/ml) (cells/ml)(U/106 cells)
4 2.0 x 106 39
2.6 x 106 378
2.6 x 106 886
2.0 x 1o6 ~sooo
These data are each from a separate run and are not intended
to demonstrate any correlation to inducer concentration.
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EXAMPLE 6.
GROWTH OF CELLS
ON IMPROVED MICROCARRIERS
FOR THE PURPOSE OF PRODUCING VIRUSES
The ability of microcarrier grown cells to produce
a virus is described here. Primary and secondary chicken
embryo fibroblasts were grown in microcarrier culture
according to the procedure described in Example 2 with the
primary cells grown at 10 grams/liter and the secondary at
- 10 5 grams/liter microcarrier concentration. To initiate virus
production, growth medium was removed, and the cultures were
washed twice with 100 ml of serum free DMEM. Infection of
cells with Sindbis virus took place in 50 ml of DMEM supple-
mented with 1% calf serum, 2% tryptose phosphate broth, and
enough Sindbis virus to equal an MOI (multiplicity of
infection) of 0.05.
The virus was harvested 24 hours after infection,
by collecting culture broth, clarifying at low centrifuga-
tion, and freezing the supernatant. Virus production was
assayed by plaque formation in a field of secondary chicken
fibroblasts. The results of infecting these microcarrier
cultures were:
All Concentration
For Production
Cell Type (cells/ml) (PFU/ml) PFU/cell
Secondary 4.0 x 106 8.4 x 109 2,100
Primary 1.4 x 106 2.3 x 101 16,000
Primary 6.0 x 106 2.6 x 101 5,000
Virus production was also established for the
following virus/cell on microcarrier combinations: Polio/
WI-38; Moloney MuLV/Cl-l mouse and VSV/chick embryo
fibroblasts.
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EXAMPLE 7.
COMPARATIVE GROWTH OF CELLS IN ROLLER BOTTLES
AND WITH IM2ROVED MICROCARRIERS FOR THE PURPOSE
OF PRODUCING MURINE LEUKEMIA VIRUS PROVIRAL DNA
The reverse-transcribed DNA of Moloney leukemia
virus (M-MULV) after infection of JLS-V9 cells, a mouse
bone marrow line, was studied.
One technique involved growing cells in roller
bottles. Cells were grown in roller bottle culture, the
medium removed, and virus inoculum introduced into the
bottles. Shortly thereafter, the cultures were fed with
fresh medium, and 8-16 hours later extracted for eventual
purification of viral DNA. The cultures were washed with
fresh buffer and the cell lysed with a solution containing
the detergent sodium dodecylsulfate. Subsequent cooling of
the lysate and addition of salt to one molar caused co-
precipitation of the detergent with high molecular weight
DNA. The low molecular weight DNA remaining in the super-
natant could then be deproteinized and concentrated for
further analysis.
A 50-roller bottle culture contained about 10~
cells. These were infected with about one-liter of viral
inoculum titering at 3 x 106 plaque-forming units per ml.
This resulted in a nominal multiplicity of infection of 1-3
and the infected cells yielded 5-20 nanograms of virus-
specific DNA.
A simpler procedure was developed employing
improved microcarriers according to this invention. A
culture containing 10 grams of beads in one liter of growth
medium was used. Upon reaching confluence, the 10 cells
on the beadQ were infected by allowing the beads to settle
out and replacing the medium with 1 liter of virus inoculum.
For extraction, the cells on the beads were washed with
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buffer and then placed in the SDS containing buffer.
After co-precipitation of the high molecular weight DNA
with the detergent, the precipitate together with the
beads were centrifuged out and a supernatant extracted
for further analysis. The yield of viral DNA was compara-
ble.to that obtained in roller bottle culture and the labor
involved was 5-10% of that required by roller bottle
culture.
EXAMPLE 8.
IMPROVED MICROCARRIER PRODUCTION WITH
DIMETHYLAMrNOETHYL CHARGE GROUPS
A suitable microcarrier was produced by binding
an alternate exchange moiety to the dextran matrix utilized
in Example 1. Dimethylaminoethyl groups (DMAE) were bound
to a dextran matrix by the following procedure: 1 gm of
dextran beads (Pharmacia G-50), 50-75 pm in diameter, dry,
was added to 10 ml of distilled water and the beads were
allowed to swell. An aqueous solution containing 0.01
moles of dimethylaminoethyl-ch~oride:chloride (Sigma
Chemical Co.) and 0.015 moles of sodium hydroxide was
formed in a 10 ml volume. This aqueous solution was
added to the swollen dextran beads and this suspension was
then agitated vigorously for one hour at 60C. After
reaction, the bead mass was titrated as in Example 1. This
reaction binds 1.0 meq of dimethylaminoethyl to the dextran
mass. To produce microcarriers of greater degrees of sub-
stitution, the above reaction was carried out, and the bead
mass washed thoroughly with water. With excess water
filtered off, the bead mass was weighed so as to determine
the amount of water being retained by the bead mass. To
this bead mass was added the appropriate amount of fresh
reagents ti.e., DMAE-CL:CL, and NaOH) so that the final
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concentration of DMAE, and NaOH in these succeeding reaction
mixtures were identical to those initially used.
In this manner, a series of microcarriers were
prepared at 1.0, 2.0, 2.5 and 3.5 meq DMA~/gm unreacted
dextran. Cells (~EL 299) were grown in microcarrier culture
(S gm/l) with these microcarriers according to the proced-
ures in Example 2. The results are tabulated in the follow-
ing table:
Degree of
Substitution
(meq/gm) Cell Spreading Net Growth
1. 0
2.0
2.5 + +
3.2 +
As expected, cell growth is related to the degree
of substitution with charge carrying groups. At too high a
degree of substitution, no cell growth occurs, although
attachment and spreading takes place. At too low a degree
of substitution, cell adhesionito the surface is not suffi-
cient to allow proper spreading and growth.
EXAMPLE 9.
IMPROVED MICROCARRIERS HAVING POSITIVELY
CHARGED PHOSPHONIUM GROUPS ,~ -
Improved microcarriers were also prepared using
non-amine exchange groups as follows. One gram of dry
dextran beads were prepared and swollen with water as in
Example 1. To the swollen beads were added 5 ml of a
saturated aqueous solution of triethyl-(ethyl-bromide)-
h i ( ) 4~5~C ~S d 5 1 f 3
tion of sodium hy~roxide. This slurry was reacted at 65C.
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A series of microcarriers were prepared at 1.1, 1.7 and 2.9
meq/gm. The microcarriers at 1.1 meq/gm were prepared by
reaction at the above conditions for 4 minutes. The 1.7
meq/gm microcarrier was reacted for 1 hour, and the 2.9
meq/gm microcarriers were reacted successively 3 times as
described in Example 7. A microcarrier cell culture at
5 gm/liter was established for each of these carriers with
a continuous cell type, JLS-V9 and compared to this cell's
growth on improved DEAE-microcarriers prepared as in
Example 3. The results are tabulated in the following
table.
DEAE
Cell Attachment
meq/gram and SpreadingNet Growth
0.9 + +
1.7 + +
3.8 . +
TEP
1.1 + +
1.7 + _ +
2.9 +
It will be recognized by those skilled in the art
that there are certain equivalents to the specific tech-
niques, materials, etc., described herein, and these are
considered to be part of this invention and are intended to
be covered by the following claims. Additionally, while
most of the description herein has been limited to the use
of the improved microcarriers for growth of anchorage-
dependent cells, they can also be used, of course, for the
growth of other cell types.
