Microcarrier Culture: Applications in Biologicals

Production and Cell Biology

CHARLES L. CRESPI, TOSHIKO IMAMURA, PHAIK-MOO1

LEONG, ROBERT J. FLEISCHAKER, HENRI BRUNENGRABER,
and WILLIAM G. THILLY, Department of Nutrition and Food
Science, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139, and DONALD J. GIARD, Cell Culture Center,
Massachusetts Institute of Technology, Cambridge, Massachusetts
02139

Summary
This report reviews recent progress in microcarrier technology at the Massachusetts
Institute of Technology. This progress includes new understanding of the nutritional needs
of high-cell-density microcarrier cultures, the demonstration of cell growth on microcarriers
in a hormone-supplemented, serum-free medium, and the continuous cell propagation of
epithelial cell types in microcarrier culture by medium modification that permits efficient
bead-to-bead transfer. Also, a technique for obtaining large quantities of mitotic cells by
selective detachment from microcarriers is reported. Finally, recent progress in interferon
production from human foreskin fibroblasts grown on microcarriers is outlined: Our im-
provements in the interferon “superinduction” process have increased the interferon yield
per cell fivefold to tenfold.

INTRODUCTION

The development of microcarrier technology has made it possible to

culture economically large numbers of anchorage-dependent cells in a
single vessel. The primary conception of this technology was by van
Wezel,’ who used Sephadex DEAE-50 as the first model. Further studies
were required before it was recognized that the parameter of surface
charge density had a narrow range of values which permitted both rapid
cell attachment and growth at high cell Now optimally
charged microcarriers are commercially available and have been used o
produce viruses4 and interferon.’
The theoretical and technical aspects of microcarrier culture have been
outlined in several recent articles .6-8 These articles provide a discussion
of the important parameters in microcarrier cultures. These parameters
are cell inoculum preparation, initial cell density, initial microcarrier con-

Biotechnology and Bioengineering, Vol. XXIII, Pp. 2673-2689 (1981)

0 1981 John Wiley & Sons, Inc. CCC OOo6-3592/8 1/122673-17$01.70
2614 CRESPI E T AL.

TABLE 1
Cell Types Successfully Grown on Microcarriers
Cell type Reference
Normal diploid cell strains
Human embryonic lung
MRCJ f,h
WI-38 b,f
HEL-299 a
Flow 2002 f
IMR-90 f
Human foreskin cells (FS-4) b,c,d,f,h
Human embryonic kidney f
Chicken embryo fibroblasts ahf
Mouse embryo fibroblasts (several strains) f
Chimpanzee liver fibroblasts f
Rat embryo fibroblasts f
Rat glial cells f
Feline lung fibroblasts f
Rabbit embryo fibroblasts f
Secondary monkey kidney cells b,f
Primary cells
Monkey kidney cells f
Dog kidney cells f
Rabbit kidney cells f
Mouse embryo fibroblasts f
Mouse macrophages f
Rat pancreas cells f
Rat hepatocytes f
Chicken embryo fibroblasts b,fj
Rat pituitary cells f
Amniotic fluid cells f
Established and transformed cell lines
Mouse fibroblasts
sc-1 f
3T3 f
CL-1 b
3T6 f
Normal rat kidney (NRK) f
Chinese hamster ovary b ,e ,f,h
Chinese hamster lung f
Baby hamster kidney (BHK-21) f
Chimpanzee embryo lung f
African green monkey kidney
Vero f&i
cv-I fLi

BSC-I, BGM f
Mouse L cells (various sublines) f
HeLa e,f
Mouse macrophage cell line f
Transformed dog kidney f
Sarcoma virus transformed rat kidney f
Sarcoma virus transformed mouse fibroblasts f
 MICROCARRIER CULTURE APPLICATIONS 2675

TABLE I (continued from previous page)

Human glioma f
Human osteosarcoma f
Madin-Darby canine kidney (MDCK) f, h
KB cells (human oral carcinoma cells) f
Rhesus monkey kidney (LLC-MK2) fhj
McCoy cells (human synovial fluid) f
Human thyroid carcinoma f
Human rhabdomyosarcoma I
Rat muscle-derived fibroblasts f
Rabbit cornea cells f
a Reference 2.
Reference 4.
' Reference 5 .
Reference 9.
Reference 25.
Reference 7 (detailed cell growth data not provided).
Reference 8.
This report.
i Reference 26.
J Reference 27.

centration, culture volume, serum supplement, nutrient depletion, stirring

speed, and microcarrier physical characteristics.
Currently, over 50 different cell types have been grown successfully
on microcarriers (see Table I). Our experience has been that any an-
chorage-dependent cell type which can be propagated sucessfully in roller
bottle culture can also be grown on microcarriers.
Our purpose here is to review the progress made in the various MIT
laboratories during the past year. This progress includes:
1) the development of a technique for continuous cell propagation using
bead-to-bead transfer;
2) improved media useful for high-density microcarrier culture;
3) the growth of Madin-Darby canine kidney cells in a hormone-sup-
plemented, serum-free medium;
4) the development of a technique for selective detachment of mitotic
cells from microcarriers; and
5) improvements in interferon production utilizing human fibroblasts
grown on microcarriers.

MATERIALS AND METHODS

Cell Lines
Human diploid foreskin (FS-4) cells were obtained from Jan Vileek
(New York University School of Medicine, New York). The maintenance
2616 CRESPI ET AL.

of stock FS-4 cell cultures is described elsewhere.’ CHO-K1 cells (ATCC

CCL 61), Madin-Darby canine kidney (MDCK) cells (ATCC CCL 34),
MRC-5 cells (ATCC CCL 1711, and LLC-MK2 cells (ATCC CCL 7) were
obtained from Flow Laboratories (McLean, VA). All cell types were
maintained in 150-cm2 plastic tissue culture flasks (Costar Plastics, Cam-
bridge, MA). CHO-K1 cells were grown in RPMI medium 1640 supple-
mented with 15% fetal calf serum (FCS). LLC-MK2, MRC-5, and MDCK
cells were grown in Dulbecco-modified Eagle medium (DMEM) supple-
mented with 10% FCS. (Media and FCS were obtained from either Flow
Laboratories or Gibco Laboratories, Grand Island, NY).

Materials
Microcarriers were either prepared using the method of Levine et al.’
or, more recently, obtained from Flow Laboratories under the trade name
Superbeads. Fructose, maltose, glucose, transferrin, insulin, triiodothy-
ronine, prostaglandin E l , hydrocortisone, dibutyrylcyclic AMP, cycloh-
examide, actinomycin D, and colcemid were obtained from Sigma Chem-
icals, St. Louis, MO. Polyinosinic-polycytidylic acid was obtained from
PL Biochemical Co. (Milwaukee, WI). Human plasma protein (Plasma-
mate) was obtained from Daly Hospital Supply (Lynnfield, MA). All
enzymes used in metabolite assays were obtained from
Beohringer-Manheim (Indianapolis, IN).

Cell Propagation on Microcarriers

The initiation of microcarrier cultures has been described previously.2
Briefly, microcarriers were suspended in phosphate-buffered saline at a
concentration of 10 mg/mL and sterilized in glass bottles by autoclaving.
The microcarriers were then dispensed into sterile centrifuge tubes
washed once with cell culture medium and aliquotted into spinner flasks
(Wilbur Scientific, Boston, MA). Due to the high oxygen requirements
of microcarrier cell cultures, spinner flasks were equipped with a thistle
tube to permit gas exchange between the head space and the atmosphere.
Final microcarrier concentration was routinely 5 mg/mL for FS-4 and
CHO-K1 cells and 2.5 mg/mL for LLC-MK2, MRC-5, and‘MDCK cells,
except that we used 1 mg/mL for MDCK cells in serum-free, hormone-
supplemented medium.

Microcarrier Quality Control

All microcarrier lots used in this report were pretested for the support
of MRC-5 cell growth. MRC-5 cells were chosen because they are very
sensitive to adverse culture conditions, and thus provide a reliable test
of microcarrier quality, Microcarrier cultures were prepared as described
earlier; 2.5 mg/mL of microcarriers were suspended in DMEM containing
 MICROCARRIER CULTURE APPLICATIONS 2677

L,
,,,
,,,
0 1 2 3 4 5 6
DAYS

Fig. 1. Growth curve of MRC-5 human fibroblasts on microcarriers. MRC-5 cells were
grown using the optimum microcarrier concentration (2.5 mg/mL) and glutamine concen-
tration (4mM) in DMEM supplemented with 10% FCS. Each point represents the mean of
ten independent determinations (ten independent lots of microcarriers were used, four pre-
pared at MIT and six obtained from Flow Laboratories under the trade name Superbeads).
Confidence limits represent 95% confidence limits on the mean.

4mM glutamine (optimal conditions of MRC-5 cell growth on microcar-

riers) and 3 x lo5 freshly trypsinized cells/mL were added. Cell number
was monitored daily. Microcarrier cultures were fed by replacement of
50% of the medium on days 2 and 4. Microcarrier lots meeting the fol-
lowing criteria were chosen for use:
1) MRC-5 cells attached rapidly (less than 2 h) and quantitatively
(greater than 95% attached) to the microcarrier surfaces;
2) no growth lag was observed (an increase in cell number during the
first day of culture);
3) rapid cell growth reached 1 x lo6 cells/mL; and
4) cells covered all (greater than 95%) microcarriers evenly.
Figure 1 is the averaged growth curve of ten independent lots of mi-
crocarriers tested with MRC-5 cells. Cell growth was highly reproducible
from lot to lot (error bars represent 95% confidence limits).

Cells Counts
Cells in microcarrier cultures were enumerated by counting cell nuclei
with a modification of the technique of Sanford et al.,'' as described by
van Wezel. '
2678 CRESPI ET AL.

Interferon Production
The superinduction procedure used was a modification of the method
reported by Have11 and VilCek.’’ Cells were grown to confluency on
microcarriers in volumes from 100 to 5000 mL (a cell density of lo6 cells/
mL reached between days 6 and 8 of growth) and then primed. The cells
were primed by washing twice with DMEM without serum supplement
and then adding DMEM plus 0.5% human plasma protein (HPP) and 50
units/mL of interferon. The cultures were incubated for 16 h at 37°C. The
cells were then washed and resuspended in DMEM containing 50 pg/mL
of poly(I):poly(C) and 10 pg/mL cyclohexamide. After incubation for 4
h at 34°C actinomycin D was added to a final concentration of 1 pg/mL.
After an additional 2 h incubation, the medium was removed and the cells
were washed twice with DMEM. DMEM containing 0.5% HPP was then
added and the cultures incubated for 1 h at 37°C and then shifted to 30°C
for 24-48 h. All media were prewarmed to the appropriate temperature.
Culture fluids were clarified by centrifugation at 2000g for 10 min and
either assayed immediately or frozen at - 70°C until assayed. The inter-
feron assay is described elsewhere.”

CONTINUOUS CELL PROPAGATION USING BEAD-TO-BEAD

TRANSFER

Microcarrier technology has been extended by the finding that two

mammalian epithelial cell lines can be subcultured continuously by simple
bead-to-bead transfer in normal medium commonly used for suspension
cell culture. Data are reported which show that the hamster ovary line
CHO-K1 and the monkey kidney line LLC-MK2 can be subcultured by
adding fresh microcarriers to the stirred suspension culture. Thirteen
generations of continuous exponential growth were demonstrated with
two such subcultures for the CHO-Kl cells and with four such subcultures
for the LLC-MK2 cells. Cell generation times were unchanged by this
subculturing approach as compared to the standard subculturing proce-
dure using trypsin to remove cells from surfaces.
This demonstration may lead to application in areas in which the ability
to propagate mammalian epithelial cells would be useful, i.e., production
of biological and viral vaccines. In particular, bead-to-bead transfer offers
a facile method for scale-up of microcarrier cultures; a 1-L microcarrier
culture could ostensibly be scaled up to 1000 L via three dilutions of 1 : 10
with fresh medium and microcarriers. This technique avoids the necessity
of trypsinizing thousands of roller bottles to inoculate a large fermentor
and the inherent risk of culture contamination from this operation.
The determination of the cell growth parameters in microcarrier culture
was the first step in developing bead-to-bead transfer. CHO-Kl cells were
grown using 5-mg/mL microcarriers in RPMI medium 1640 (a reduced
 MICROCARRIER CULTURE APPLICATIONS 2679

0 2 4 6 8 10 12 14 16 18
DAYS

Fig. 2. Continuous growth of CHO-Kl(-) and LLC-MK2 (-) cells on microcarriers

using bead-to-bead transfer. The arrow indicates dilution of a high-density culture into fresh
medium and microcaniers.

calcium medium) supplemented with 15% fetal calf serum (FCS); when
the cell density reached 2 x lo6 cells/mL, 50% of the growth medium
was replaced. Under these cell growth conditions a cell inoculum of 3
x lo5 cells/mL attached to the microcarrier surfaces within 2 h and grew
with a 17-h doubling time to a final cell concentrhtion of 5 x lo6 cells/
mL after 4 days. LLC-MK2 cells were grown using 2.5-mg/mL micro-
carriers in 50% minimum essential medium without calcium (MEM- ) and
50% RPMI 1640 supplemented with 10% FCS. Under these cell growth
conditions a cell inoculum of 2 x lo5 cells/mL attached to the microcarrier
surfaces within 2 h and grew with a 27-h doubling time to a final cell
concentration of 1.6 x lo6 cells/mL after 4 days.
After determining the cell growth parameters in microcarrier culture,
the feasibility of subculturing using bead-to-bead transfer was tested.
Bead-to-bead transfer was achieved by dilution of a nearly confluent
(50430% of available surface occupied) microcarrier culture with 80-95%
v/v fresh growth medium containing fresh microcarriers. After dilution,
2680 CRESPI ET AL.

cells appeared to grow on the “old” microcarriers. One day following

dilution, however, 2-5 cells were present on the “new” microcarriers.
The cells on the new microcarriers grew and eventually covered all avail-
able microcarrier surfaces. Throughout this process an exponential
growth rate was observed.
To test the feasibility of continuous cell growth using bead-to-bead
transfer in microcarrier culture, a CHO-K1 microcarrier culture was in-
oculated and diluted 1 :20 every three days; an LLC-MK2 microcarrier
culture was inoculated and diluted 1 : 5 every four days. The results are
presented in Figure 2. By using sequential dilutions, 13 generations of
exponential growth were observed (a 10,000-fold increase in cell number).
Cell doubling times were unchanged using this bead-to-bead transfer tech-
nique as compared to a traditional technique using trypsin to remove cells
from surfaces. The CHO-K1 cells grew with a 16-h doubling time and the
LLC-MK2 cells grew with a 31-h doubling time.
Cell propagation in standard suspension cell culture medium formulae
(reduced calcium media) was apparently sufficient to permit bead-to-bead
transfer for the CHO-KI and LLC-MK2 cell lines. Whether this simple
protocol will be generally applicable to other epithelial cells in culture is
not yet clear. Regarding fibroblastic cells, only chick embryo fibroblasts
have been studied in terms of bead-to-bead transfer, and while preliminary
results are encouraging, a generally useful bead-to-bead transfer system
for fibroblasts has yet to be devised.

NUTRITIONAL ASPECTS OF HIGH-DENSITY MICROCARRIER

CULTURES

In cell cultures grown in the presence of glucose, glycolysis generates

high concentrations of lactate and a concomitant drop in culture pH. This
phenomenon is particularly important in microcarrier cultures because
the high cell densities usually encountered can radidly decrease the cul-
ture pH to suboptimal levels. As early as 1958, Eagle observed better
utilization of f r ~ c t o s e than
’ ~ glucose. Rheinwald and GreenI4 noted that
the slow glucose feeding via maltose ameliorated the pH decrease, and
recently another group’5 has noted the good utilization characteristics of
fructose in cultured cells. Those who study cell cultures at high cell
densities which are obtained in microcarrier culture are aware particularly
of the limitation imposed by pH changes. We have examined thus the
potential of fructose in microcarrier culture of MDCK cells, and reex-
amined the potential of maltose as a means to achieve high cell densities
without significant pH decline.
Microcarrier cultures were prepared using optimally charged micro-
carriers (2.5-mg/mL final concentration) suspended in Dulbecco’s mod-
ification of Eagle’s medium (DMEM) supplemented with 10% FCS ac-
 MICROCARRIER CULTURE APPLICATIONS 268 1

t
6.5 -
1
0 I 2 3 4 5
T i m e (days1

Fig. 3. pH profile during culture of MDCK cells with (-) glucose (20mM), (A-A)
fructose (20mM), and (-) maltose (5mM).

-I
f /
Time (days)

Fig. 4. Lactate production by MDCK cells with (-) glucose (20mM), (A-A) fructose
(ZOmM), and (-) maltose (5mM).
2682 CRESPI ET AL.

;L
2100

ig245

1235

4225

0 I 2 3 4 5
Time (days)

Fig. 5. Redox potential of the cytosolic NADH/NAD+ couple in MDCK cells as meas-
wed by the lactate/pyruvate ratio in the culture medium. Conditions are identical to those
in Figs. 2 and 3.

cording to the protocol outlined by Levine, Wang, and T h i l l ~ .The~

concentration of cells in the culture was determined daily. Aliquots (4
mL) of the medium were sampled daily for the determination of the pH
and metabolite concentrations. Cells were separated from the medium16
which was then deproteinized with perchloric acid (final concentration
3%). The acid extract was neutralized with a solution of 0.5N trietha-
nolamine in 2N KOH. The neutral extract was analyzed for pyruvate"
and lactate'' by standard enzymatic assays. The redox potential of the
NADH/NAD+ couple in the cells was estimated from the lactate/pyruvate
ratio in the medium.
In a series of preliminary experiments we determined that the maximum
growth of MDCK cells in high-density microcarrier culture (2.5 mg/mL
microcarriers suspended in DMEM supplemented with 10% FCS without
medium replacement) was achieved with either 20mM glucose, 20mM
fructose, or 5mM maltose. (When maltose was the carbohydrate source,
maltase activity in FCS was sufficient to hydrolyze 5 m M maltose in 5
days in cell-free medium. The hydrolysis was linear and corresponding
maltase activity was 5.9 mU/mL FCS.) When either carbohydrate source
was used, a cell inoculum of 5 x lo5 cells/mL attached rapidly to the
microcarrier surfaces (less than 2 h) and grew with a 20-24-h doubling
time reaching a maximum cell density of (2.5-3) x lo6 cells/mL after 2-3
 MICROCARRIER CULTURE APPLICATIONS 2683

days. The p H profiles of the various culture media are shown in Figure
3. The glucose-containing culture pH dropped below 7.0 by the second
day, while pH remained above 7.2 in both maltose- and fructose-con-
taining cultures.
The production of lactate by MDCK cells was reduced by a factor of
3 or 4 when glucose was substituted by maltose or fructose (Fig. 4). The
redox potential of the cytosolic NADH/NAD+ couple was measured by
the lactate/pyruvate ratio in the culture medium is shown in Figure 5. It
can be inferred from published data that the physiological range for the
lactate/pyruvate ratio is 6-15.19,20 At the start of the culture, the ratio
was more oxidized than physiological as it was imposed by the formulation
of the medium. In the culture started with 20mM glucose, the lactate/
pyruvate ratio increased to nonphysiological levels after 2 days. In con-
trast, in the presence of fructose or maltose, the ratio remained within
physiological limits up to days 3 and 5 , respectively.
We intend to extend these nutritional studies to developing a medium
suitable for single-batch microcarrier culture to high cell densities. To
this end we are systematically investigating all nutrients which may be
limiting cell growth.

GROWTH OF MDCK CELLS ON MICROCARRIERS IN A

HORMONE-SUPPLEMENTED, SERUM-FREE MEDIUM

Cells in culture show a high dependence on serum for growth. It has

been proposed that the main function of serum is to provide complexes
of hormones.2' Recently, a hormone-supplemented, serum-free medium
has been developed in Sato's laboratory2*for the growth of MDCK cells
in stationary culture. In this report, the serum requirement was substituted
with transferrin, insulin, triiodothyronine, prostaglandin El , and hydro-
cortisone (Table 11) in a growth medium consisting of a 50 :50 mixture of
DMEM and Ham's F-12.
We have met with success in growing stationary cultures of MDCK
cells without serum using the hormone-supplemented medium developed

TABLE I1
Composition of Serum-Free Medium
Supplement Stationary culture" Microcarrier culture
Insulin 5 pg/mL 25 p&nL
Transfenin 5 CLg/mL 25 pg/mL
Hydrocortisone 50nM 50nM
Triiodothyronine 5PM 5PM
Prostaglandin El 25 ng/mL 25 ng/mL
Dibutyryl CAMP Not required 42 & n L
SeO: ~ lOpM lOpM
a Reference 22.
2684 CRESPI ET AL.

3
2o t 1
10 ---
7 --
5 -
X
Y
3 -
-
E
L 2-
a
a

I -

0.7 -
I I 1 I I

0 I 2 3 4 5
DAYS

Fig. 6. Growth of MDCK cells in a 50:50 mixture of DMEM and Ham’s F12 supple-
mented with (-) 10% horse serum, (-) hormone supplement developed at MIT (serum-
free), and (-) hormone supplement developed for stationary culture (serum-free), with
I-mg/mL microcarriers.

in Sato’s laboratory. Unfortunately, this formulation did not support the

growth of MDCK cells on microcarriers (Fig. 6).
We have found that an increase in the concentration of transferrin and
insulin and the addition of dibutyrylcyclic AMP permitted the growth of
MDCK cells on microcarriers (1 mg/mL) (see Table I1 and Fig. 6). The
cell doubling time observed in this serum-free microcarrier culture was
equivalent to the doubling time observed when cells were grown in the
same medium supplemented with 10% horse serum. However, an initial
lag period of 1 day was seen in the growth of the cells in the hormone-
supplemented, serum-free medium.
The demonstration of MDCK cell growth in a serum-free, hormone-
supplemented medium now permits studies concerning regulation of cell
growth without serum interference. This demonstration also permits the
continuous growth of cells under strictly controlled serum-free conditions
without any exogenous proteins (such as trypsin or trypsin inhibitors)
through bead-to-bead transfer.

MITOTIC DETACHMENT FROM MICROCARRIERS

We are now able to describe a method for collecting large quantities

of mitotic cells from a population of CHO-K1 cells growing on micro-
carriers in suspension culture. Exponentially growing cells were treated
 MICROCARRIER CULTURE APPLICATIONS 2685

for 2.5 h with colcemid, and the mitotic cells were detached by increasing
the stirring speed. A yield of up to 6% of the total population can be
obtained using this method. Of the cells collected, 85-95% were visibly
arrested in metaphase as determined by microscopic inspection.
A microcarrier culture (in a 250-mL spinner bottle equipped with a 4.5-
cm magnetic stir bar available from Wilbur Scientific, Boston, MA) con-
taining S-mg/mL microcarriers suspended in 100 mL of RPMI 1640 sup-
plemented with 15% FCS was inoculated with I x lo8 CHO-K1 cells.
The culture was stirred at 60 rpm and the cells allowed to grow for 20 h
reaching a cell density of (2-2.5) x lo6 cells/mL; at this time the cells
are still in exponential growth. Loosely bound cells were shaken free by
increasing the stirring speed to 150 rpm for 10 min. The stirring was then
stopped and the microcarriers were allowed to settle for approximately
5 min. The supernatant containing the detached cells was removed by
aspiration. The microcarrier-attached cells were washed twice with pre-
warmed medium; each time a brief, gentle stir was applied. After the
second wash, the microcarriers were suspended in 100 mL of growth
medium and colcemid was added to a final concentration of 30 ng/mL.
The spinner culture was then stirred for 2.5 h at 60 rpm. After incubation,
the microcarriers were washed once to remove the colcemid and the
volurtte adjusted to 100 mL. The culture was then stirred at 150 rpm for
10 min, the microcarriers allowed to settle, and the supernatant containing
the mitotic cells collected. The yield of this protocol with CHO-Kl cells
was 4-6% of the total population [(8-12)x lo6 cells/100 mL spinner].
The mitotic index of the collected cells was 85-95%.
Plates containing mitotic cells were incubated at 37°C; every 2 h one
plate was removed, washed once with phosphate-buffered saline, and
trypsinized. The trypsinized cells were counted on a hemacytometer.
Figure 7 is a typical growth curve for synchronous cells. The initially
mitotic cells undergo the first round of division within 2 h. The cell number
then remains constant until 15 h after plating. Between 15 and 20 h the
cells undergo a second division typically to 1.75-1.8 of the initial plateau
value. The growth curve gives an Engelberg synchronization i n d e ~ * ~ * * ~
of approximately 90% for the first division and 60% for the second di-
vision. The median cell cycle time for the synchronous population was
16 h, which is similar to that of an exponentially growing culture.
After the cell count was determined, the modal volume of the population
was determined using a Coulter model ZB1 particle counter. Figure 7 is
a graph of the modal volume at different times after plating. After 3 h the
modal volume was unimodal at a volume of 255 pm3. The volume nearly
doubled to 500 pm3 after 11 h. After 15 h, the distribution was bimodal,
with peaks at 260 and 500 pm3. This corresponds to the time of the second
cell division. After 19 h the peak was again unimodal with a volume of
265 pm3.
This technique for mitotic selection does have one drawback: The col-
2686 CRESPI ET AL.

/ GI / s / G2.M / GI

500 1
E
w 15 - - 450 a
I- W
a
a
J
0
- 400 5
\ .A
v) - 350 ’
0
j
w
10
_J
a
u
8- - 300
5
6 - - 250

I 3 5 7 9 11 13 15 17 19 21 23
HOURS AFTER PLATING

Fig. 7. (M) Growth curve of mitotic CHO-Kl cells after plating in tissue culture dishes.
(-) Modal volume of the selected population at intervals after plating. The time of S
phase was determined by 3H-thymidine incorporation.

cemid treatment appears to render the selected population more sensitive

to excessive shear. Cell viability, as monitored by Trypan blue exclusion,
dropped from 90 to 75% when colcemid-treated cells were pipetted vig-
orously ten times through a standard bore 10-mL pipette.

PRODUCTION OF INTERFERON UTILIZING HUMAN FIBROBLAST

GROWN ON MICROCARRIERS

The success of this project has depended on two developments: the

successful growth of human foreskin fibroblasts (FS-4) on microcarriers
and the improvement of the interferon induction process.
The following conditions were found to be essential for the successful
culture of FS-4 cells on microcarriers:
1) the microcarrier culture inoculum must consist of cells harvested
from an exponential growth phase culture;
2) the inoculum density must exceed 3 x lo5 cells/mL when using a
microcarrier concentration of 5 mg/mL (i.e., an average of 10 cells/mi-
crocarrier); and
3) the concentration of fetal calf serum supplement should be reduced
from 10% v/v to 5%.
Figure 8 is a typical growth curve of FS-4 cells in microcamer culture.
This particular set of data was obtained from a 5-L run. Cell growth was
 MICROCARRIER CULTURE APPLICATIONS 2687

measured both by enumeration of cell nuclei and by measuring the oxygen

demand of the culture (expressed as oxygen uptake rate).
Important progress has been made in the optimization of the interferon
induction process.' We have reexamined and modified a number of steps
in the interferon superinduction process. During superinduction the cells
are first exposed to a viral-like agent (specifically double-stranded RNA).
The cells are then treated with specific antimetabolites. Finally, the cells
are washed and transferred to a production medium. The effects of the
antimetabolites are to enhance and prolong the interferon production. We
have found that the following parameters are important in the superin-
duction process :
1) The cells should be induced just upon entering stationary growth
phase.
2) Priming is beneficial. (Priming is the exposure of the cells to a small
amount of interferon prior to induction.)
3) The concentration and length of exposure to the antimetabolites is
important.
4) The temperature profile during the induction and production periods
can be manipulated successfully to enhance greatly the length of inter-
feron production.
When this work was begun, interferon yields were inconsistent and
seldom over lo4 units/106 cells. At present, as shown in Table 111, we are
able to obtain interferon yields consistently between 5 x lo4 and lo5
units/106 cells (at a cell density of 1 x lo6 cells/mL in microcarrier cul-

"i
10

7 07

1,,
, I 1.01
30 60 90 120 150 180

Hours
Fig. 8. Growth curve of FS-4 human fibroblasts on microcarriers: both (-) number
of cell nuclei and (0-0)oxygen uptake rate are plotted.
2688 CRESPI E T AL.

TABLE I11
Rate of Interferon Production Using Temperature Shift and Extended
Production Period
Rate of interferon production
(units/106 cells 24 h)
Experiment No. First 24 h ( x 10') Second 24 h ( x lo3)
100 mL
I 54 ND"
2 43.4 ND
3 18 44
4 22 25.5
5 28 28.5
5L
1 24 24
2 65 45
a ND-not determined.

ture). Furthermore, we have grown and induced successfully FS-4 cells

to produce interferon on a 5-L scale; we see no reason why production
could not be performed on a still larger scale.
In summary, we have developed a means to produce high-titered low-
cost interferon. The advantages of this method are:
1) Scale-up should not be a problem;
2) the economics are quite favorable-our average materials cost for
the 5-L runs was $2.50/106 units of interferon (work already in progress
indicates this price can be reduced fourfold to fivefold);
3) our product is a fully glycosylated and complete protein. This factor
is likely to be important with regard to the activity, in vivo stability, and
antigenicity of the compound; and
4) finally, since the interferon is excreted into the medium, purification
is relatively simple and does not involve complex separations.

The development of bead-to-bead transfer and serum-free cell growth, and the study of
the nutritional aspects of microcarrier cultures were supported by a gift from Flow General,
Inc. The authors would also like t o acknowledge grant No. NIH-5T32-ES-07020-3 from the
National Institute of Environmental Health Sciences which provided a traineeship for
Charles L. Crespi and National Cancer Institute grant No. 1-T32-C809258-01 which provided
a traineeship for Robert Fleischaker. The interferon research was supported by a grant from
the Louis and Rosa Strauss Memorial Fund.

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Accepted for Publication February 23, 198 1