Page 8

NaOH to the ice-cold collagen-acetic acid solution. The
final collagen concentration in this mixture is 2.75 mg/ml,
and the dilution factors are such that the final concentrations
of salts and other nutrients are nearly identical to culture
medium. In a 35-mm culture dish, 1.5 ml of this collagen
solution provides an adequate substratum for flounder renal
epithelial cell attachment. It is important to note that while
the collagen concentration is critical, the volume of collagen
solution added (and therefore the total collagen content of
the dish) is equally important. The collagen mixture must be
left undisturbed for about 30 min at room temperature for
complete gelation. The gels are then thoroughly rinsed and
equilibrated with normal culture medium.

By the same methods described above, fish skin colla-
gen is prepared from flounder skin which has been scraped
free of scales and loosely adherent tissues, and then minced.
A fundamental difference in the fish and rattail collagen is
the effect of temperature on gelation. Maintenance of rattail
collagen plating mixture on ice very effectively delays
gelation. In contrast, the winter flounder fish skin collagen
plating mixture will gel rapidly even if ice cold.

The “contraction” of collagen by the epithelium is a
biphasic process. The first stage of contraction occurs at a
relatively rapid rate, while the second stage is much slower.
Gel collagen concentration was inversely proportional to the
rate and extent of contraction and seemed to influence both
the fast and slow phases. Changes in the fast phase of
contraction were obvious since initiation of contraction was
delayed as the gel collagen concentration was increased. In
fact, in our hands, it was possible to inhibit contraction
almost totally by using gels that were highly concentrated.
Changes in the slow phase of contraction were apparent as
well since the proportionality between collagen concentra-
tion and gel size was maintained throughout the duration of
the culture period. The observation that collagen concentra-
tion influences renal epithelium contraction of collagen gels
may be relevant to other cell culture systems. It is also true
that the use of different cell seeding densities influences
contraction.

Contractable (undenatured) collagen gel substrata per-
mit flounder renal cells to assume a semblance of their
normal columnar shape in culture. When the cells are first
isolated they are sphere-shaped. On plastic, they flatten
when attached. This flattened appearance persists even in
confluent monolayers. Transmission electron micrographs
have shown that the cells assume their normal columnar
shape and tight junctions on contracted collagen.

Cells which were cultured on collagen substrata of
differing contractability exhibited markedly different
transepithelial electrical properties. On a totally
noncontractable substratum--glutaraldehyde-fixed collagen
gels--flounder renal epithelium consistently exhibited lower
transepithelial electrical potentials, lower total charge trans-
fer (short-circuit currents), and higher transepithelial elec-
trical resistances than identical cells cultured on contractable
collagen gels. Cells cultured on high-density, native colla-
gen gels which had contracted to a negligible degree also

exhibited low potentials and short-circuit currents, while
transepithelial resistance was intermediate to values ob-
served for contracted gels and fixed gels.

Whether serum is present or replaced by specific hor-
mones and growth factors, the culture medium is important
in defining the differentiated characteristics expressed. The
expression of tissue-level functions is obviously profoundly
affected by certain culture medium constituents. However,
certain effects may be insidious in that survival may be
promoted while differentiation is prevented. A striking
example of this can be seen in the effect of the aminoglycoside
antibiotic, streptomycin, on flounder proximal tubule pri-
mary cultures (Dickman and Renfro 1990). Streptomycinis
a commonly used prophylactic in culture media. Unfortu-
nately, it is also a fairly potent nephrotoxin (Humes et al.
1982). Inthe flounder renal system, 100 pg/ml streptomycin
caused total loss of all measured transport functions. The
renal monolayer remained viable and readily contracted
collagen gels. Ciliary activity was still apparent at the light
microscope level. Thus, the source of this toxic effect was
difficult to isolate due to the long-term persistence of a
viable monolayer. Ultrastructurally, an almost total loss of
apical microvilla was seen. A toxic effect on transepithelial
transport could be detected with as little as 2 ug/ml strepto-
mycin present, and the cells had not recovered even 6 days
after total removal of the antibiotic.

Another aspect of the culture medium which must be
considered is the potential for certain nutritive constituents
toinduce regulatory-type modification of variably expressed
transport phenomena. Thus, especially in tissues such as
renal proximal tubules which are subject to regulatory
controls, a particular set of culture conditions must be
regarded as only one of possibly many which may support
varying differentiated states.

In the case of renal proximal tubule epithelium, almost
all of the culture medium constituents are potentially trans-
portable or transformable substrates. Variations in any of
these ligand levels may significantly modulate a specific
transport system. We have seen several examples of the
effects of altered transportable substrate concentration on
transport capabilities of flounder primary monolayer cul-
tures. The tissue responds rapidly to medium phosphate
concentration. Secretory phosphate flux was stimulated by
increasing the medium phosphate concentration. The re-
sponse to substrate change took less than 90 min. Thus, a
possible important regulation of renal phosphate excretion
may be phosphate itself.

In most cases, it is not clear how changes in levels of
transportable substrate in culture medium cause changes in
transepithelial transport processes. Factors which should be
considered include the effect of the medium substrate on
cellular metabolism, the possibility that the substrate may be
accumulated inside the cells and promote mediated self-
exchange, the possibility that a substrate may cause minor
injury and induction of the stress response, and the possibil-
ity that receptor-mediated signaling through cellular second
messengers is induced by certain substrates. These are


Page 25

APPENDIX C

WORKSHOP AGENDA

Topic

Speaker

Introduction: Historical Perspectives
Needs, Uses, and Benefits

Breaking the Barriers to Continuous
Long-term Cell Propagation

Homeothermic Cell Cultures
Poikilothermic Cell Cultures
Invertebrate Cell Cultures
Insects
Arachnids
Freshwater Mollusks
Other Species
Insights and Recent Advances Toward
Understanding Continuous Culture of
Marine Invertebrate Cells: Open
Discussion
Crustaceans
Sponges
Other Marine Invertebrates

Mollusks/Comments

Workshop Summary: Synthesis and
the Future

A. Machii and A. Rosenfield (Conveners)
C. Reinisch

A. Machii (Moderator)

L. Hayflick

J.L. Renfro

F. Hink
T. Kurtti
C. Bayne
S.N. Chen

A. Rosenfield (Moderator)

D. Fadool

S. Pomponi

From the audience
L. Ellis

R. Goodwin (Moderator) and C. Bayne

Ye U. 5. GOVERNMENT PRINTING OFFICE: 1993--700-026--80000


Page 2

Machii, A.; Komada, S.; Awaji, M. 1989. Balanced salt
solution for pearl oyster cell culture. Venus Jpn. J.
Malacol. 48: 55.

Machii, A., Wada, K.T.; Awaji, M.; Nakamura, H.K.;
Townsley, S.J. 1988. Some characters of cells of the mid-
gut gland and chytrids from primary cultures of the prawn

Penaeus japonicus. In: Kuroda, Y.; Kurstak, E.;
Maramorosch, K., eds. Invertebrate and fish tissue cul-
ture. Tokyo: Japanese Scientific Societies Press; p. 11-14.
Yano, Y.;Machii, A. 1975. Aminoacid composition of dark
brown secretion formed in organ culture of mantle tissue
of the pearl oyster. Bull. Jpn. Soc. Sci. Fish. 41: 1083.

MARINE INVERTEBRATE CELL CULTURE RESEARCH IN NORTH AMERICA

Aaron Rosentield
Oxford Cooperative Laboratory
National Marine Fisheries Service
Oxford, Maryland 21654

MOLLUSCAN DISEASE STUDIES

It was about 35 yr ago that several marine-oriented
laboratories in North America undertook concerted efforts
to develop methods for the long-term cultivation and main-
tenance of marine molluscan cells and tissues. Using then-
existing vertebrate cell culture approaches, the major objec-
tive of these efforts was to find in vitro systems that would
allow for the isolation and identification of cytozoic patho-
genic agents suspected as the cause for the devastating
mortalities of eastern oyster, Crassostrea virginica, popula-
tions taking place along the eastern coasts of the United
States and Canada: Chesapeake Bay, Delaware Bay, Gulf of
Mexico, and Canadian maritime provinces. These areas at
that time were the major oyster harvesting “grounds” of
North America, with Chesapeake Bay representing one of
the largest commercial oyster-producing ecosystems of the
world. Thus, there existed socioeconomic, cultural, politi-
cal, and intellectually compelling forces to find the etiologic
agent(s) responsible for the observed epizootics. Further-
more, cell culture and other in vitro cultivation methods
were expected to be used to good advantage for conducting
epizootiological studies that would provide information on
the spread and distribution of aquatic animal diseases, and
on the prevalence, incidence, and intensity of infection of
those diseases. It would naturally follow that if these
cultivation methods could be developed successfully, then
other biochemical and biophysical information would be
acquired that would prove useful for studying: (1) host cell
and pathogen gene action; (2) disease replication, transmis-
sion, infectivity, and virulence; and (3) activation-inactiva-
tion of immune mechanisms. Of course, a major consider-
ation of shellfish resource managers was to apply the infor-
mation gained to devise disease prevention, control, and
eradication measures. Today in North America, molluscan
cell culture efforts continue, but in vitro systems for cultiva-
tion of molluscan pathogens have not yet been achieved.

MARINE CRUSTACEAN DISEASE
STUDIES

During the mid- to late 1960s, and continuing even until
today, disease studies on marine crustaceans have become
of equal if not of greater importance than oyster or other
molluscan disease studies. This is largely due tothe frequent
occurrences of gill and shell lesions, discolorations, and
extensive unexplained mortalities of economically valuable
species of shrimps, lobsters, and crabs harvested from
commercial fishing grounds along the eastern and Gulf of
Mexico coasts of the United States. Furthermore, world-
wide interest in the rapidly expanding field of crustacean
mariculture has drawn attention to the possibility that im-
ported animals will carry infectious agents.

Several state and Federal government agencies, aca-
demic institutions, and aquaculture industry laboratories
have recognized that the impact of imported disease(s),
particularly of viral, fungal, or bacterial origin, could serve
as major deterrents to breeding, hatchery, and growout
operations, as well as to husbanding aquaculture species.
We have learned a great deal from the large body of existing
literature, as well as from practical experience (and the use
of common sense), of the dangers inherent in the transmis-
sion of communicable diseases by infectious pathogenic
agents associated with the movement of plants and animals
inhabiting terrestrial ecosystems. The pathways and mecha-
nisms for disease transmission operative in terrestrial eco-
systems act according to the same principles and apply
equally to aquatic ecosystems. Hence, the need is compel-
ling for early detection and disease control strategies in
which in vitro cell culture systems will play an important
role. As with the lack of availability of molluscan cell
culture systems, little progress in North America has been
made toward establishing a long-term, in vitro, crustacean
cell culture system useful for pathogen detection or for other
studies.


Page 12

culture--penicillin, streptomycin, and gentamicin. The de-
ficiencies in basal L15 are multifactorial as it lacks glucose,
amino acids, organic acids, vitamins, and trace minerals
known to be important for invertebrates. Based on our
knowledge of invertebrate biochemistry, we modified L15
to formulate L15B which promotes tick cell growth in low
FBS concentrations and the absence of TPB (Munderloh
and Kurtti 1989).

INOCULUM FOR PRIMARY CULTURES

Lines have been isolated from embryonic and develop-
ing adult tissues. Primary cultures from embryonic tissues
are easy to initiate by crushing eggs, while the adult tissues
must be dissected from nymphs shortly before they molt to
the imago stage.

Table 1. Selected invertebrate cell lines isolated using Leibovitz’s L15 medium
Invertebrate Cell Line Inoculum Medium Reference
Code
Nematoda
Meloidogyne incognita MI8C Egg-laying FBS; hemolymph, Manousis and
females Manduca sexta Ellar (1990)
Arachnida
Rhipicephalus appendiculatus TTC 243 Developing FBS; TPB Varma et al.
adult tissues (1975)
Insecta
Hemiptera
Triatoma infestans BTC 32 Embryonic FBS; TPB Pudney and
tissues Lanar (1977)
Diptera
Toxorhynchites amboinensis TAE 12V Embryonic FBS;TPB Munderloh et al.
tissues (1982)
Table 2. Tick cell lines banked at the University of Minnesota, Department of Entomology
Species Stage “Cell Line Reference
Code
Rhipicephalus appendiculatus developing RA 243 Varma et al. (1975)
adult
Dermacentor variabilis embryo RML-15 Yunker et al. (1981)
Rhipicephalus appendiculatus embryo. RAE 25 Kurtti et al. (1982)
Rhipicephalus sanguineus embryo RSE8 Kurtti and Munderloh (1982)
Anocentor nitens embryo ANE 58 Kurtti et al. (1983)
Boophilus microplus embryo BME 26 Munderloh and Kurtti (1989)
Dermacentor albipictus embryo DALBE 18 Witherspoon and Kurtti, unpublished*
Ixodes dammini embryo. DE 12 Munderloh and Kurtti, unpublished”

* University of Minnesota, Department of Entomology, 1980 Folwell Ave., St. Paul, MN 55108.

> Ibid.



BIBLIOGRAPHIC INFORMATION
PB93-213593

Report Nos: NOAA-TM-NMFS~F/NEC-98

Title: Marine Invertebrate Cell Culture: Breaking the Barriers. Proceedings of an
International Workshop. Held in Anaheim, California on June 16, 1991.

Date: Mar 93
Authors: A. Rosenfield.

Performing Organization: National Marine Fisheries Service, Woods Hole, MA.
Northeast Fisheries Science Center.

Type of Report and Period Covered: Technical memo.

NTIS Field/Group Codes: 47D
Price: PC AQ3/MF Agl

Availability: Available from the National Technical Information Service,
Springfield, VA. 22161

Number of Pages: 34p

Keywords: “Marine biology, “Invertebrates, “Meetings, Cultured cells, Tissue
culture, Kidney tubules, Insects, Ticks, Lobsters, Signal transduction, Mollusca,
Insects, Marine fishes, Porifera.

Abstract: The workshop concerned marine invertebrate cell and tissue culture and
was part of the World Congress on Cell and Tissue Culture cosponsored by the Tissue
Culture Association and the Japanese Tissue Culture Association and held in
Anaheim, California, during 16-26 June 1991. The workshop explored the possible
reasons for the lack of success, despite vigorous attempts over a period of more
than 58 years, to continuously grow lines of cells derived from tissues of marine
and other invertebrate species. Long-term in vitro maintenance of tissues and
primary cell cultures of some marine invertebrate species has been achieved-~and
used to good advantage. These marine invertebrate cells contain an extraordinary
diversity of genetic information and possess other properties that can be used to
good advantage for many basic and applied purposes.


Page 23

APPENDIX A

ABSTRACT: RESULTS OF TISSUE AND CELL CULTURE
OF FISH AND AQUATIC INVERTEBRATES

S.N. Chen
Department of Zoology
National Taiwan University
Taipei, Taiwan

Results on tissue and cell culture of fish and aquatic
invertebrates have been obtained by the laboratory of S.N.
Chen in the past 11 yr.

Twenty cell lines derived from warmwater fish have
been established from eel (Anguilla japonica), tilapia hybrid
(Serotherodon mossambicus x S. niloticus), top-minnow
(Gambusia patruelis), color carp (Cyprinus carpio), com-
mon carp (C. carpio), snakehead (Channa maculata), loach
(Misgurnus anguillicaudatus), and banded grouper
(Epinephelus awoara), Jarbua fish (Therapan jarbua), striped
mullet (Mugil cephalus), milkfish (Chanos chanos), and
black carp (Mylopharyngodon piceus). These cell lines
have been widely and successfully used for the study of

virology, cell physiology, and molecular biology.

Attempts on the establishment of cell culture systems
from mollusks have also been carried out. The results
showed that monolayer cultures with approximately 10
passages have been obtained from heart, mantle, and em-
bryos of oysters, Crassostrea gigas and Ostrea edulis, and
hard clams, Metrix lusoria or M. metrix.

Monolayer cultures were also established from heart,
ovary, and lymphoid tissue peripheral hemocytes of penaeid
shrimp, including Penaeus monodon, P. japonicus, and P.
penicillatus. Culture system derived from lymphoid tissue
“Oka organ” was found to be susceptible to Monodon
baculovirus in vitro.


probably not discrete responses, but the latter is potentially
highly specific. Second-messenger control of differentiated
tissue function can be profoundly influenced by the history
of physicochemical stimuli.

The development of the flounder renal proximal tubule
primary monolayer culture system (Dickman and Renfro
1986) has permitted the gathering of considerable informa-
tion about the transport mechanisms and regulation of
teleost proximal tubule. Unidirectional transport rates for
several substances which are typically handled by proximal
tubule were determined in Ussing chambers under short-
circuited conditions. As expected, in the marine renal
tubule, sulfate is normally secreted. Organicanion secretion
and glucose reabsorption are normal for proximal nephron
of almost all vertebrates and are amply expressed in this
culture system.

Page 9

REFERENCES

Dickman, K.G.; Renfro, J.L. 1986. Primary culture of
flounder renal tubule cells: transepithelial transport. Am.
J. Physiol. 251: F424-F432.

Dickman, F.G.; Renfro, J.L. 1990. Streptomycin toxicity in
primary cultures of flounder renal proximal tubule cells.
In Vitro Cell. & Dev. Biol. 26: 571-578.

Gupta, A.; Renfro, J.L. 1989. Control of phosphate trans-
port in flounder renal proximal tubule primary cultures.
Am. J. Physiol. 256: R850-R857.

Humes, H.D.; Weinberg, J.; Knauss, T. 1982. Clinical and
pathophysiologic aspects of aminoglycoside and
nephrotoxicity. Am. J. Kidney Dis. 11: 5-29.

Michaelopoulos, G.; Pitot, H. 1975. Primary culture of
parenchymal liver cells on collagen membranes. Exp.
Cell Res. 90: 70-78.

TECHNIQUES FOR INSECT CELL CULTURE

W. Fred Hink
Department of Entomology
The Ohio State University
Columbus, Ohio 43210

This is a brief review of procedures, concepts, and
results in the establishment of cell lines from four species of
insects (cotton bollworm, cabbage looper, codling moth,
and cat flea), and in the maintenance of primary cultures
from ant and honeybee venom glands.

MEDIA

The chemical composition of media (amino acids,
vitamins, sugars, organic acids, salt, etc.) is obviously of
concern and much has been written about the rationale for
formulating media by attempting to duplicate the hemo-
lymph biochemically. Other characteristics such as pH,
oxygen tension, osmotic pressure, and ion balance may need
to be optimized. In practice, however, the decision on which
medium to use is usually based on a review of the literature
and then choosing media that give favorable results with
similar species. When evaluating media and/or modifica-
tions, one must keep in mind that there are tremendous
variations in the appearance of individual primary cultures
in the same medium. This makes it imperative to use many
cultures (at least six) to evaluate each new medium.

Inour work with media for venom gland cell cultures,we
found that conditioning the medium with an insect cell line
greatly increased cell viability. Several lines from Lepidopt-

era and Diptera were tested, and one, IPLB-SF 1254, pro-
vided significantly better growth conditions. This suggests
that for other invertebrates, it may be necessary to examine
media from numerous lines. Conditioning was done by
using cultures with an initial cell density of 2x 10° cells/ml,
incubating for 72 hr, harvesting the medium, removing cells
by centrifugation, adjusting pH to the original value (7.0),
and sterilizing by filtration. Conditioned and unconditioned
media were mixed 1:1 because this combination theoreti-
cally contains the “beneficial factors” from growing cells
and added nutrients from fresh medium that might have been
utilized during the conditioning process.

We have evaluated numerous materials to promote cell
viability, longevity, and multiplication. Most were ineffec-
tive, but some were beneficial and I want to discuss them
here because others may want to test them with other
invertebrate cells.

Epidermal growth factor (EGF): A protein extracted
from mouse submaxillary glands that induces mamma-
lian epithelial cell proliferation and DNA synthesis. At
1.83 ng/ml, it stimulated cell migration from the ex-
plants and seemed to promote cell division.

Fibroblast growth factor: A protein extracted from
bovine pituitary glands that stimulates DNA synthesis


barriers themselves, their identity, and their “location”
within the cell cycle: a

a.

d.

Identities and lack of necessary growth hormones?
Try “bypass”? (Does new information on roles of
the extracellular matrix imply that attention paid to
this would be rewarding?)

Lack of essential nutrients? Screen for ability of
nutrients to improve the appearance of primaries
and delay their deterioration.

Preculture block to propagation? Modify prepara-
tion protocols.

In-culture growth inhibition? Liberate.

Where in the cell cycle is the block? Do new stage-
specific antigens in other systems provide tools: for
answering this? Following are some notes from
Hansen's discussion of factors and problems in devel-
opment of the Bge cell line:

a.

Selection of tissue source. Embryos. She tried, but
didnot exploit, “decontrolling” theembryos’ growth
by irradiation and lithium chloride prior to disrupt-
ing them.

Sterilization. Surface sterilization of egg masses
with iodine and hyamine. Primary tissues with
abundant antibiotics. But these were then washed
out; there were no antibiotics in regular media,
though there are in the Bge medium.

Cell separation. Fragments tended to reaggregate.
Pressing the trypsin/EDTA-treated fragments
through a 30-ym sieve yielded single cells and
small clumps.

Selecting and testing media. For primary cultures,
media were often based on hemolymph composi-
tion (sugars, amino acids, and salts). The success-
ful medium was among the simpler formulations.
Once a line was established, it was simple to screen
components, and to discover the utility of lactalbu-
min hydrolysate. Of various vertebrate sera tested,

Page 15

only FBS was supportive, and lot-to-lot variability
was marked. Heat inactivate; avoid hemolyzed
lots. The fetal calf serum (FCS) seemed essential
for good cell attachment, but otherwise deleteri-
ous; it was added and removed repeatedly for
primary cultures. Once the Bge cell line was
established, the medium could be modified so as to
be more readily available in other laboratories.

The formulation (Hansen 1976a) is: Schnei-
der’s + FCS, galactose, lactalbumin hydrolysate,
and antibiotics.

Hansen considered that early use of dilute
trypsin, even without passaging, was critical to her
success.

3. Other considerations gleaned from the nonmarine mol-
juscan literature:

a. Screen your lots of FBS!

b. Doyou have access to water with quality adequate
for high-performance liquid chromatography?

c. Don’tassumecells require FBS; try them in serum-
free culture.

REFERENCES

Bayne, C.J. 1981. Gastropod cells in vitro, Adv. Cell Cult.
1: 297-334,

Hansen, E.L. 1976a. A cell line from embryos of
Biomphalaria glabrata (Pulmonata): establishment and
characterization. In: Maramorosch, K., ed. Inverte-
brate tissue culture: research applications. New York:
Academic Press; p. 75-99.

Hansen, E.L. 1976b. Application of tissue culture of a
pulmonate snail to culture of larval Schistosoma
mansoni. In: Kurstak, E., Maramorosch, K., eds. Inver-
tebrate tissue culture: applications in biology, medicine
and agriculture. New York: Academic Press; p. 87-97.

Odintsova, N.A.; Khomenko, A.V. 1991. Primary cell
culture from embryos of the Japanese scallop
Mizuchopecten yessoensis (Bivalvia). Cytotechnology
6: 49-54,


multiplying and in good morphological coridition. The
critical issue is to determine when and how to’subculture.
Generally, we subculture when about 75% of the surface of
the primary is covered with attached cells. We have experi-
mented with numerous enzymes (elastase, collagenase, hya-
luronidase, pronase) and find that trypsin is best for retention
of cell viability.
Procedure for the cell line from flea tissue:

1, Use Ca’*- and Mg**-free Hanks’ phosphate-buffered
saline with additional glucose to make it iso-osmotic to
the medium + 0.002% ethylenediaminetetraacetic acid
(Hanks’ EDTA).

2. Remove medium.

3. Add 0.25% trypsin in Hanks* EDTA and incubate 10
min.

4. Remove trypsin and add fresh medium.

5. Gently rotate dish, remove medium, and add 2.0 ml
fresh again (a rinse).

6. Pipet a stream of medium over the cells to flush them
off.

7. Transfer total volume of dish (2.0 ml) to a 25-cm?
tissue-culture flask containing 3.0 ml medium.

In early subcultures, trypsin had a delayed detrimental
effect on cells observed 24-48 hr post-treatment, so it was

Page 11

absolutely necessary to rinse them before transfer to the
flask. After the line was growing well (at the 8th subculture)
and maintained in flasks, it was not necessary to rinse off the
trypsin.

Procedure for subculturing an established, firmly at-
tached cell line:

1. Remove medium.
2. Add 5.0 ml Hanks’ EDTA, swirl, and remove.
3. Add 1.2 ml 0.25% trypsin and incubate 7 min.

4. Remove trypsin, twist cap on tight, and let culture sit for
3 min.

5. Bang flask against palm of hand several times to dis-
lodge the cells.

6. Add 5.0 ml fresh medium and swirl to obtain a cell
suspension.

7. Subculture every 7 days using split ratios of 1:2.5 to
1:3.3 (2.0 ml parent into 3.0 ml fresh to 1.5 ml parent
into 3.5 ml fresh). After the 20th subculture, the split
ratio was adjusted to 1:6.25.

Others have released attached cells using physical
methods such as flushing with a strong stream of medium,
scraping, or stirring with a magnetic spin bar. We find these
methods unacceptable because they damage cell mem-
branes.

TICK CELL CULTURE TECHNOLOGY

Timothy J. Kurtti and Ulrike G. Munderloh
Department of Entomology
University of Minnesota
St. Paul, Minnesota 55108

Based on the pioneering work of Rehacek (1965),
Varma et al. (1975), Yunker and Meibos (1979), Yunker et
al, (1981), and Yunker (1987), several references describe
the establishment and maintenance of tick cell cultures
(Rehacek 1965; Kurtti and Bischer 1979; Pudney et al.
1979; Yunker and Meibos 1979; Yunker 1987). In recent
years, these techniques have led to the successful isolation
of several new tick cell lines. Using a modification of
Leibovitz’s medium, L-15B (Munderloh and Kurtti 1989),
we have been able to establish cell lines from every tick
species tried. However, the technology to isolate, at will,
lines from specific cell types has not been developed. As is
necessary for the cultivation of specialized vertebrate cells,

we expect that invertebrate hormones and growth factors
will be important in achieving this task.

MEDIA

The medium found to give the best results in the culture
of tick cells is Leibovitz’s L15 medium, which is available
fromseveral suppliers. When supplemented by FBS, tryptose
phosphate broth (TPB), and, in some reports, lactalbumin
hydrolysate, bovine serum albumin (fraction V), and TC
yeastolate, it supports a wide range of invertebrate cells
(Table 1). Antimicrobials used are standard for tissue


Page 24

ACES
BES
cAMP
EDTA
EGF
FBS
FCS
G-proteins
GTP
HEPES
IP,
MES
ROI
SSF
TGF-B
TPB

APPENDIX B

ACRONYMS

N-2-acetamido-2-aminoethanesulfonic acid
N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid
cyclic adenosine monophosphate.
ethylenediaminetetraacetic acid

epidermal growth factor

fetal bovine serum

fetal calf serum

GTP-binding proteins

guanosine triphosphate
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
inositol triphosphate
2-(N-morpholino)-ethanesulfonic acid

Teactive oxygen intermediates

sea star factor

transforming growth factor - beta

tryptose phosphate broth


Page 6

BRIEF HISTORY OF THE DEVELOPMENT OF HUMAN DIPLOID CELL STRAINS

Leonard Hayflick
Cell Biology and Aging Section
University of California, San Francisco
San Francisco, California 94121

Exactly 30 yr ago, Paul Moorhead and I described the
finite replicative capacity of normal human fibroblasts and
interpreted the phenomenon to be aging at the cell level. We
showed that when human embryonic cells are grown under
the most favorable conditions, aging and death are the
inevitable consequences after about 50 population doub-
lings. We called this the phase III phenomenon. In more
recent times, others, unaware of our work, have coined the
word apoptosis to explain the same phenomenon in vivo.

We showed that the death of cultured normal human
cells was not due to some trivial cause involving medium
components or culture conditions, but was an inherent
property of the cells themselves. The observation has since
been confirmed in hundreds of laboratories.

At the time our observation was made, the paramount
dogma in cell biology was that the failure of cells to
proliferate indefinitely in vitro must be attributable to errors
in the”art” required to keep cells dividing forever.

In describing the human diploid cell strains, we re-
ported that these strains have several interesting properties:

1. If derived from human embryos, cell strains undergo
about 50 population doublings. The potential cell yield
from 50 population doublings is about 20 million metric
tons.

2. Human diploid cells undergo a number of population
doublings inversely proportional to donor age. This
suggested to us that the finite replicative capacity of
cultured normal cells is an expression of aging at the cell
level. This notion received considerable experimental
support in subsequent years and became the basis for
the field of cell aging that we named cytogerontology.

3. If derived from normal tissue, cell strains have the
diploid karyotype, and--unlike immortal cell lines--
normal cell strains are incapable of replication in sus-
pension culture. We did not give this property a name,
but it is now called anchorage dependence.

4. Human cell strains will not produce tumors when
inoculated into the hamster cheek pouch or even when
directly inoculated into terminal human cancer patients.

5. Human diploid cell strains can be cryogenically pre-
served, When, for example, our first widely distributed
normal human diploid cell strain, WI-38, which we
developed in 1962, is preserved at a particular doubling
level and then reconstituted, the number of doublings

remaining is equivalent to 50 minus the number of
doublings spent prior to preservation. The cells have an
extraordinary memory and “remember” at what dou-
bling level they were preserved even after 29 yr of
continuous storage in liquid nitrogen. WI-38 has been
preserved longer than any other normal human or
animal cell population.

Asa result of this characterization, we suggested that all
cultured animal and human cells be classified into three

groups:

1. The primary cell culture, derived from intact tissue and
undergoing no subcultivations, is Group I.

2. The cell strain which has a finite capacity to replicate,
does not produce tumors when inoculated into experi-
mental animals, has the karyology of the tissue of
origin, and is anchorage dependent, is Group II.

3. The cell line which is a population of immortal cells,
may produce tumors when inoculated into laboratory
animals, does not have the karyology of the tissue of
origin, and is usually anchorage independent, is Group
Ill. These are transformed cells.

In respect to marine invertebrate cells that do not seem
to transform, this could be a blessing in disguise. Although
there are reasons why immortal marine invertebrate cells
might be useful, in most cases I doubt that this would be so.
My objection to the use of cell lines was stated earlier; that
is, these cells are not normal. Assuming that marine inver-
tebrate cell lines would also be abnormal, their use would be
limited to research in which normal cells would not be
required. Then, the question becomes: Of what value are
cells that do not resemble any class of cells in the anatomy
of the original donor?

But this apparent dilemma does have a solution. As-
suming that some marine invertebrate cell types can be
cultured, and that they behave in general like vertebrate
cells, one would expect them to undergo several population
doublings before they age and die, just as do vertebrate cell
strains. Even if the number of population doublings that
these strains undergo is as few as 20, it would result in the
possibility of cryopreserving well over 1,000 ampules at the
10th population doubling, and of the contents of each
reconstituted ampule yielding 1,000-fold more cells. Thus,
any marine invertebrate cell strain that behaved in this way
could be available for use by many laboratories and for a




Malewicz, B.; Kumar, V.V.; Johnson, R.G. 1990. Lipids in
gap junction assembly and function. Lipids 25: 419-427.

Massague, J. 1990. Transforming growth factor-a, amodel
for membrane-anchored growth factors. J. Biol. Chem.
265: 21393-21396.

Merrill, A.H. 1989. Lipid modulators of cell function. Nutr.
Rev. 47: 161-169,

Meyer, R.; Malewicz, B.,; Baumann, W.J. 1990. Increased
gap junction assembly between cultured cells upon cho-
lesterol supplementation. J. Cell Sci. 96: 231-238.

Meyer, R.A.; Lampe, P.D.; Malewicz, B. 1991. Enhanced
gap junction formation with LDL and apolipoprotein B.
Exp. Cell Res. 196: 72-81.

Mitsuhashi, J. 1989. Nutritional requirements of insect cells
in vitro, In: Mitsuhashi, J., ed. Invertebrate cell system
applications. Vol. 1. Boca Raton, FL: CRC Press; p. 3-20.

Moses, H.L.; Barnard, J.; Bascom, C.C. 1991. Control of
proliferation by transforming growth factors. In: Bellvé,
A.R.; Vogel, H.J., eds. Molecular mechanisms in cellular
growth and differentiation. San Diego, CA: Academic
Press; p. 121-128.

Munderloh, U.G.; Kurtti, T.J. 1989. Formulation of me-
dium for tick cell culture. Exp. & Appl. Acarol. 7: 219-
229.

Page 21

Nottebohm, F. 1985. Neuronal replacement in adulthood.
Ann. N.Y. Acad. Sci. 457: 143-161.

Nottebohm, F.; Alvarez-Buylla, A.; Cynx, J. 1990. Song
learning in birds; the relation between perception and
production. Philos. Trans. R. Soc. Lond. B Biol. Sci. 329:
115-124,

Ridgeway, R.L., Syed, N.L.; Lukowiak, K. 1991. Nerve
growth factor induces sprouting of specific neurons of the
snail, Lymnaea stagnalis. J. Neurobiol. 22: 377-390.

Sutton, G.C.; Russell, N.J.; Quinn, P.J. 1991. The effect of
salinity on the phase behavior of total lipid extracts and
binary mixtures of the major phospholipids isolated from
a moderately halophilic eubacterium. Biochim. Biophys.
Acta 1061: 235-246.

Thomas, U.; Speicher, S.A.; Knust, E. 1991. The Dros-
ophila gene Serrate encodes an EGF-like transmembrane
protein with a complex expression pattern in embryos and
wing discs. Development 111: 749-761.

Thompson, S.N.; Mejia-Scales, V.; Borchardt, D.B. 1991,
Physiologic studies of snail-schistosome interactions and
potential for improvement of in vitro culture of schisto-
somes. In Vitro Cell. & Dev. Biol. 27A: 497-504.

BREAKING THE BARRIERS TO CONTINUOUS PROPAGATION
OF MOLLUSCAN CELLS: A SUMMARY

Christopher J. Bayne
Department of Zoology
Oregon State University
Corvallis, Oregon 97331-2914

Let me share with you the status of research we have
been doing with Dr. David Barnes and colleagues at Oregon
State University in attempts to develop cell culture systems
for Schistosoma mansoni. One year after beginning this
project, it is possible to obtain cultures routinely with a
variety of cell types and tissue fragments derived from
schistosomules and sporocysts. The cultures remain viable
for several weeks, but after the first week or so they invari-
ably begin deteriorating. Cells that initially are distributed
over the culture surface come together in groups under some
conditions, and such groups appear to be potential sites for
cell division. However, no cultures have yet yielded evi-
dence of sustainable propagation.

On several occasions we refer to the protocols used by
Dr. Eder Hansen in her successful attempts to establish a cell
line from Biomphalaria glabrata, a tropical freshwater
snail. Hansen used embryos as the source of her cells for
primary cultures. She used high concentrations of a number
of antibiotics to achieve sterile material for primary cultures.
Cells were liberated by use of trypsin-EDTA and nylon
mesh screens. Once healthy-looking, primary cultures were

subjected to periods with and without FBS, and cultures
were trypsinized without transfer to new culture containers.

A number of questions were addressed in the handout
prepared for these proceedings. These questions included
the qualities desired in the target species and life-cycle stage
selected as a source of cells, and considerations such as how
completely one should digest primary fragments to yield
cells for culture. The marine sources of tissues for culture
by those in the workshop should not present any particular
problem; the reticence of nonarthropod invertebrate cells to
propagate in culture has not been restricted to marine spe-
cies. Also, the high osmolar values desired in media allow
scope for the inclusion of a great variety of media compo-
nents (not necessarily a good thing!) and the possibility of
providing healthy levels of nutrients. This consideration
may appear more reasonable in light of the very low osmolar
values sometimes forced on those trying to culture cells and
tissues from freshwater species, some of which have blood
osmolar values as low as 100 mOsm.

It was pointed out that mechanical disruption of em-
bryos and other sources of cells for primary culture may


Page 18

methods unreliable. A precise quantification method is
needed due to the slow and currently unpredictable re-
sponses of the sponge cells. Chromatographic techniques
are used to quantify the presence and production of second-
ary metabolites.

While some sponges from a variety of habitats seem
amenable to the same culture methods, many require adjust-
ments at each step of the procedure. Each species presents
its own challenges in terms of contaminants, cell diversity,

and cell size.

Current research efforts focus on culture optimization
for long-term maintenance, growth, and/or metabolite pro-
duction. Though the problems of controlling contamination
and sustaining cultures in a variety of media have largely
been solved, some central problems remain. These include
the role of microbial symbionts, the tendency of some cells
to terminally differentiate, slow growth, and the presently
limited life span of the cultures.

IMPEDIMENTS TO THE CULTURE OF NONFIBROBLASTOID CELLS
FROM INSECTS AND OTHER INVERTEBRATES, AND THEIR RESOLUTION

Ronald H. Goodwin
Rangeland Insect Laboratory
Agricultural Research Service
Bozeman, Montana 59717

SUMMARY

Few demonstrable nonfibroblastoid cell types have
been cultured from any eukaryotic invertebrate tissues.
Most insect cell lines demonstrate no traceable differenti-
ated functions or consistent morphologies. Outside the
Insecta, primary cell or organ culture maintenance has rarely
given subcultures of any cell type and almost no finite or
continuous cell lines. The lack of progress in this research
may be related to an inappropriate comparison of inverte-
brate cell culture requirements with the culture requirements
of transformed vertebrate cells.

INTRODUCTION

The fact that some insect cell types can be continuously
cultured in certain vertebrate cell culture media has sup-
ported the notion that invertebrate cellular nutritional re-
quirements are not basically different from vertebrate cellu-
lar requirements. However, the general continued failure to
establish cell lines from other invertebrate groups suggests
that we really donot know very much about invertebrate cell
physiology. While we have inadequate criteria at present to
decide whether existing insect cell lines are transformed or
not (Hansen 1976), it is only cellular transformation that has
permitted the isolation of the majority of vertebrate cell
lines. The scientific literature describes rather startling
differences between the response of transformed and normal
vertebrate cells in culture to specific supplementations,
particularly in serum-free media, which have been increas-
ingly recognized as a basic requirement for nontransformed
cell types. The hormonal milieu of serum has been shown
to be growth preventing for vertebrate epithelial cellsas well

as other types, and this relationship may hold as well for
many invertebrate cell types (Goodwin 1991). The complex
and fastidious nature of normal eukaryotic cells in culture
prevents us from extrapolating the in vivo concept of essen-
tial nutrients to the situation presented by these cells, tissues,
and organs without significant modifications. The problems
in culturing unusual (nonfibroblastoid) invertebrate cell
types should, therefore, be measured against similar prob-
lems now coming to light in the culture of nontransformed
vertebrate cells.

DISCUSSION

Normal cellular growth or differentiation requirements
are controlled in part through intracellular connections at the
cell membrane and/or the extracellular matrix levels
(Goodwin 1991). Cells in culture that maintain some
intracellular membrane and/or matrix connections are often
enabled to grow and may also respond to (hormonal or
growth factor) commands to express tissue-specific func-
tions in vitro. The scientific literature has provided many
examples from both in vitro and in vivo experimentation of
the control of membrane protein activity and pathogen
invasion activities by specific plasma membrane lipids
(Merrill 1989; Goodwin 1991). The orientation, position-
ing, number, and activity of growth factor and hormone
receptors are controlled in part through their association
with certain membrane sphingolipids, phospholipids, and
sterols that are asymmetrically positioned in the plasma
membrane and other internal cytoplasmic and nuclear mem-
branes. A number of these phospholipids have also been
shown to demonstrate specific fatty acid species asymmetry
in a tissue-specific manner (Goodwin 1991).


1

NOAA Technical Memorandum NMFS-F/NEC-98

pe BOS ie,

This TM series is used for documentation and timely communication of preliminary results,
interim reports, or special purpose information, and has not undergone external scientific
review.

Marine Invertebrate Cell Culture:
Breaking the Barriers

Proceedings of an International Workshop,
16 June 1991, Anaheim, California

Aaron Rosenfield, Editor
Oxford Cooperative Lab., National Marine Fisheries Serv., Oxford, MD 21654

U. S. DEPARTMENT OF COMMERCE
Ronald H. Brown, Secretary
National Oceanic and Atmospheric Administration
Diana Josephson, Acting Administrator
National Marine Fisheries Service
Nancy Foster, Acting Assistant Administrator for Fisheries
Northeast Region
Northeast Fisheries Science Center
Woods Hole, Massachusetts

March 1993




significant number of studies.

If abnormal immortal cell lines are useful for some
purposes, one might explore the possibility of purposefully
transforming marine invertebrate cell strains by the same
means used for some cultured vertebrate cells--that is, use
transforming conditions such as oncogenic viruses, chemi-

Page 7

cal carcinogens, and radiation. If indigenous marine inver-
tebrate oncogenic viruses are not known, this may not be
important because most viral transformations made to occur
in vertebrate cells succeed when the oncogenic virus is not
indigenous to the animal species whose cells are to be
transformed.

TISSUE CULTURE TECHNIQUES FOR STUDY OF TRANSEPITHELIAL TRANSPORT
BY TELEOST RENAL TUBULE

J. Larry Renfro
Department of Physiology and Neurobiology
University of Connecticut
Storrs, Connecticut 06269-3042

The following is a brief review of tissue culture meth-
ods considered important for maintenance of tissue-level
differentiation in renal proximal tubule epithelium of winter
flounder, Pleuronectes americanus. The focus is on proce-
dures we have found necessary to produce fully functional
winter flounder renal proximal tubule primary monolayer
cultures. ‘

The value of fewer distinctly functioning segment types
in marine teleosts such as winter flounder is that once the
renal tubules are separated from the surrounding hematopoi-
etic tissue, one has a relatively cléan population of proximal
tubule epithelial cells and may. avoid certain preparative
steps designed to isolate a particular cell population. Thus,
choice of animal (winter flounder) eliminates one-to-sev-
eral steps in the preparation of renal proximal tubule primary
cultures since the proximal epithelium dominates, and cell
separation techniques such as differential or gradient cen-
trifugation are unnecessary.

Our objective has been to maintain differentiation while
reorganizing the tissue from a tubule structure to monolayer
form to provide a system from which definitive information
about transepithelial transport may be more conveniently
extracted. Because of the intimate relationship between
epithelial structure and function, the establishment of mor-
phological polarity in culture is prerequisite for the expres-
sion of net vectorial transport by cultured monolayers.

The study of transepithelial transport by cultured epi-

. thelia necessitates the use of a removable porous culture
support so that cultures can be mounted in Ussing chambers.
We have used native, i.e., neither denatured nor glutaralde-
hyde-fixed (cross-linked), collagen gels. Fixed or denatured
collagen cannot be contracted by any tissue. Unlike most
filter supports, collagen gels are relatively transparent, so
that the course of culture development can be viewed with
ease microscopically. Finally, the contractability of colla-
gen gels has been shown to influence differentiation of
cultured cells, and, most importantly, allows the cells to
assume a more natural cell shape in culture.

We have found that the most gentle way of isolating and
dispersing flounder renal tubule cellsis by cold trypsinization.
In this preparation (Dickman and Renfro 1986, modifica-
tions in Gupta and Renfro 1989), the kidneys are perfused
and teased apart in modified M199. The extrarenal tissues
are removed by incubating the tubules in Ca- and Mg-free
balanced salt solution with 0.2% trypsin at 22°C for 45 min
on an orbital shaker. Tubule fragments are separated from
nontubule tissues on 20-ym Nitex nylon mesh. The epithe-
lial cells are released from these fragments by 3 days of cold
trypsinization at 5°C. The cold trypsinization method was
originally used by others to prevent tryptic action while
allowing trypsin to penetrate tissues thoroughly. Subse-
quent exposure to 37°C would then produce much faster
tissue dispersion. The prolonged exposure of the poikilo-
thermic flounder renal cells to 5°C is tolerated well, and the
trypsin activity at this suboptimal temperature is very gentle.
The major advantage of this method is that ample time can
be provided for trypsin to penetrate the tough basement
membrane surrounding each nephron while minimizing
enzymatic damage to the cells. Released cells are collected
through 50-ym Nitex mesh, washed, pelleted, suspended in
modified M199 with 10% flounder serum, and plated to
confluency on native rattail or fish skin collagen.

The preparation of rattail collagen for primary hepato-
cyte cultures was described by Michaelopoulos and Pitot
(1975). The collagen preparation we have used for the
flounder renal epithelium is very similar; however, as noted,
fish skin collagen also serves as an effective matrix substra-
tum. Rattail tendons, finely teased, are sterilized with
several washes of 95% ethanol, rinsed in sterile 0.1% acetic
acid, and incubated in the latter for 2 days at 10°C. In our
hands, sterilization of the collagen with ultraviolet light
partially denatured the collagen and usually rendered it
uncontractable. Gelation of the dissolved collagen is initi-
ated by addition of 10-fold concentrated M199 culture
medium and a very basic solution of N-2-hydroxy-
ethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and



Preface

TABLE OF CONTENTS

Presentations

Marine Invertebrate Tissue Culture Research in Japan .....
Akira Machii

Marine Invertebrate Cell Culture Research in North America .
Aaron Rosenfield

Models To Illustrate the Needs, Uses, and Benefits of Marine
Invertebrate Cell Cultures
Carol L. Reinisch

Brief History of the Development of Human Diploid Cell Strains
Leonard Hayflick

Tissue Culture Techniques for Study of Transepithelial Transport by
Teleost Renal Tubule ...
J. Larry Renfro
Techniques for Insect Cell Culture ....
W. Fred Hink

Tick Cell Culture Technology ....
Timothy J. Kurtti and Ulrike G. Munderloh

Breaking the Barriers to Continuous Long-term Cell Propagation:
Freshwater Mollusks.....
Christopher J. Bayne
Cultured Lobster Olfactory Neurons: Utility in Signal
Transduction Studies...
D.A. Fadool, W.C. Michel, and B.W. Ache
Marine Sponge Cell Culture ...
S.A. Pomponi, R. Willoughby, and J.E. Armstrong

Summaries

Impediments to the Culture of Nonfibroblastoid Cells from Insects
and Other Invertebrates, and Their Resolution ....
Ronald H. Goodwin

Breaking the Barriers to Continuous Propagation of Molluscan Cell

A Summary ...
Christopher J. Bayne

Appendices

A. Abstract: Results of Tissue and Cell Culture of Fish and
Aquatic Invertebrates ...
S.N. Chen

Page iii


CELL LINE CHARACTERISTICS

The cell lines banked in liquid nitrogen in our laboratory
at the University of Minnesota are listed in Table 2.

Initial growth of tick cells in primary cultures and early
subcultures is slow, and several weeks to months may elapse
between transfers. After 1-2 yr of adaptation to in vitro
conditions during 3-5 transfers, young lines can generally be
subcultured at a split ratio of 1:3 or 1:5 every 2-4 wk.
Continuous lines are transferred more frequently or at higher
dilutions, and have population doubling times of 3-5 days.

Lines vary in cell morphology, but generally one type
predominates. The majority of cells are diploid, but aneu-
ploid and tetraploid cells have been observed. The isozymes
isocitrate dehydrogenase, malic enzyme, phospho-
glucoisomerase, and phosphoglucomutase of one line (TTC
243) have been examined (Brown and Knudson 1982). We
are comparing the isozymes of several different tick cell
lines by isoelectric focusing gel electrophoresis, and find
lactate and malate dehydrogenases to be useful enzyme
systems to distinguish between lines and confirm their
species identity.

REFERENCES

Brown, S.E.; Knudson, D.L. 1982. Characterization of
invertebrate cell lines. IV. Isozyme analyses of dipteran
and acarine cell lines. In Vitro 18: 347-350.

Kurtti, T.J.; Bischer, G. 1979. Trends in tick cell culture.
In: Maramorosch, K.; Hirumi, H., eds. Practical tissue
culture applications. New York: Academic Press; p.
351-371.

Kurtti, T.J.; Munderloh, U.G. 1982. Tick cell culture:
characteristics, growth requirements, and applications
to parasitology. In: Maramorosch, K.; Mitsuhashi, J.,
eds. Invertebrate cell culture applications. New York:
Academic Press; p. 195-232.

Kurtti, T.J., Munderloh, U.G.; Samish, M. 1982. Effect of
medium supplements on tickcells in culture. J. Parasitol.
68: 930-935.

Page 13

Kurtti, T.J.; Munderloh, U.G.; Stiller, D. 1983. The
interaction of Babesiacaballi with tickcells. J. Invertebr.
Pathol. 42: 334-343.

Kurtti, T.J.; Munderloh, U.G.; Ahlstrand, G.G. 1988. Tick
cell culture in vector research. Adv. Dis. Vector Res. 5:
87-109.

Manousis, T.; Ellar, D.J. 1990. Establishment of two cell
lines from the nematode Meloidogyne incognita
(Tylenchida: Meloidogynidae). In Vitro Cell. & Dev.
Biol, 26: 1105-1114.

Munderloh, U.G.; Kurtti, T.J. 1989. Formulation of me-
dium for tick cell culture. Exp. & Appl. Acarol. 7:219-
229.

Munderloh, U.G.; Kurtti, T.J.; Maramorosch, K. 1982.
Anopheles stephensi and Toxorhynchites amboinensis:
aseptic rearing of mosquito larvae on cultured cells. J.
Parasitol. 68: 1085-1091.

Pudney, M.; Lanar, D. 1977. Establishment and character-
ization of a cell line (BTC-32) from the triatomine bug,
Triatoma infestans (Klug) (Hemiptera: Reduviidae).
Ann. Trop. Med. Parasitol. 71: 109-118.

Pudney, M.;, Varma, M.G.R.; Leake, C.J. 1979. Establish-
ment of cell lines from ixodid ticks. Tissue Cult. Assoc.
Man. 5: 1003-1007.

Rehacek, J. 1965. Preparation of tissue cultures from the
tick Hyalomma dromedarii. J. Med. Entomol. 2: 161-
164.

Varma, M.G.R.; Pudney, M.; Leake, C.J. 1975. The
establishment of three cell lines from the tick
Rhipicephalus appendiculatus (Acari: Ixodidae) and
their infection with some arboviruses. J. Med. Entomol.
11: 698-706.

Yunker, C.E.; Cory, J.; Meibos, H. 1981. Continuous cell
lines from embryonic tissues of ticks (Acari: Ixodidae).
In Vitro 17: 139-142.

Yunker, C.E. 1987. Preparation and maintenance of arthro-
pod cell cultures: Acari, with emphasis on ticks. In:
Yunker, C.E., ed. Arboviruses in arthropod cells in
vitro. Boca Raton, FL: CRC Press; p. 35-51.

Yunker, C.E.; Meibos, H. 1979. Culture of embryonic tick
cells (Acari: Ixodidae). Tissue Cult. Assoc. Man. 5:
1015-1017.

BREAKING THE BARRIERS TO CONTINUOUS LONG-TERM CELL PROPAGATION:
FRESHWATER MOLLUSKS

Christopher J. Bayne
Department of Zoology
Oregon State University
Corvallis, Oregon 97331-2914

The following questions and answers reflect the per-
spectives I have developed in the course of efforts to
establish cell lines from freshwater mollusks and, more
recently in collaboration with Dr. David Barnes, cell culture

systems for the helminth Schistosoma mansoni. They have
been formulated in light of those procedures which led Eder
Hansen to establish the only presently available molluscan
cell line (Bge; ATCC No. CRL 1494), and of the marine


ae me

| titi fil ‘in

mre

NOAA Technical Memorandum NMFS-F/NEC-98

Marine Invertebrate Cell Culture:
Breaking the Barriers

Proceedings of an International Workshop,
16 June 1991, Anaheim, California

U. S. DEPARTMENT OF COMMERCE
National Oceanic and Atmospheric Administration
National Marine Fisheries Service
Northeast Region
Northeast Fisheries Science Center
Woods Hole, Massachusetts

March 1993

Reprodced by:
‘National Techcial fsformation Service
US. Depanent

Springheta, VA 22161

th

\
/


Page vi

Division of the Tissue Culture Association for their encour-
agement and endorsement of the workshop. Mr. Bill
Momberger, Executive Director, and his staff of the Tissue
Culture Association helped immeasurably by making ac-
commodations to fulfill last-minute requests for unpredicted
needs. Mrs. Jane Keller of the NMFS Oxford Cooperative
Laboratory has our gratitude for her role in typing and
assistance in copy editing these proceedings. Mrs. Karen

Hayman, also of the NMFS Oxford Cooperative Labora-
tory, was particularly helpful to the workshop organizers by
giving special attention to matters pertaining to correspon-
dence and program development.

A. Rosenfield (Chair)
C. Reinisch
Workshop Organization Committee


Pagev

PREFACE!

These proceedings are the result of a workshop jointly
convened by the Tissue Culture Association's Invertebrate
Division and the Society for Invertebrate Pathology:The
workshop concerned marine invertebrate cell and tissue
culture and was part of the World Congress on Cell and
Tissue Culture cosponsored by the Tissue Culture Associa-
tion and the Japanese Tissue Culture Association and heldin
Anaheim, California, during 16-20 June 1991. The work-
shop explored the possible reasons for the lack of success,
despite vigorous attempts over a period of more than 50 yr,
to continuously grow lines of cells derived from tissues of
marine and other invertebrate species. Long-term in vitro
maintenance of tissues and primary cell cultures of some
marine invertebrate,species has been achieved--and used to
good advantage! {ENevertheless, the need to develop addi-
tional “immortal” and replicating cell culture systems for
long-term in vitro propagation of marine invertebrate cell
lines is still compelling. Development of such systems will
allow for the cells/| to be cloned, characterized, stored,
retrieved, transportéd, and shared with cooperating labora-
tories. Their use will help ensure uniformity and reproduc-
ibility of results. Furthermore, the systems will provide for
consistent production of an enormous variety of unique cells
from many phyla.~These marine invertebrate cells contain
an extraordinary diversity of genetic information and pos-
sess other properties that can be used to good advantage for
many basic and applied purposes. -ganelt usefulness in the
various fields of biotechnology and genetic engineering
ultimately will be staggering. Itis al8o obvious that achiev-
ing a breakthrough in the long-term continuous culture of
marine invertebrate cells would go far in helping to resolve
questions in comparative pathobiology, research. They
would be particularly useful in a parallel way, as cell lines
have been useful with homeothermic, poikilothermic verte-
brate, and insect cell culture systems, for the controlled
propagation and in-depth study of infectious agents (such as
viruses) whose growth requirements are so fastidious that
they require living host-specific cells in which to , replicate.

In addition to invertebrate pathologists, a number of
tissue culture experts were invited to the worksHop who
were successful in the propagation of difficul-to-grow cells

\

originating from a wide array of homeothermic and poikilo-
thermic taxa, including mammals, fish, insects, and arach-
nids. These experts critiqued past failures. They discussed
and advised those interested and involved with marine
invertebrate cell culture on how to improve their chances for
success to continuously cultivate cells over several genera-
tions.

Further studies were also presented by investigators
currently active in research involving marine invertebrate
cell biology and related fields. These reports provided
additional insights for the propagation and uses of marine
invertebrate cell culture. A summary report of the workshop
was also presented extemporaneously by Dr. Ronald
Goodwin. His interpretations of some of the information
presented by the speakers drew upon his research with
growth factors and membrane physiology. Dr. Goodwin
was also kind enough to furnish a copy of a report prepared
for inclusion in the proceedings of the Eighth International
Conference on Invertebrate and Fish Tissue Culture that was
held in conjunction with the 1991 World Congress on Cell
and Tissue Culture. It is included as part of the summary to
this workshop. Dr. Christopher Bayne also provided some
additional reflections after the meeting on the report that he
presented at the workshop. His later comments are also
included as part of this workshop summary.

From this workshop, it is expected that many innovative
ideas will be generated and may lead tosome approaches for
producing long-term culture of cell lines from marine inver-
tebrates. Itis also anticipated that the workshop will produce
agreements for cooperative actions calling for a worldwide
multilaboratory assault aimed toward the development of
cell lines from several candidate species of marine inverte-
brates, for example from crustaceans, mollusks, echino-
derms, annelids, coelenterates, and even, perhaps, primitive
chordates.?

In closing, we thank the National Oceanic and Atmo-
spheric Administration’s Office of Ocean Pollution and
National Marine Fisheries Service (NMFS) for supporting
the workshop and publication of these proceedings. Special
thanks are extended to the Council of the Society for
Invertebrate Pathology and to the officers of the Invertebrate

1 The mention of trade names does not imply endorsement by the National Marine Fisheries Service.

‘The workof Professor S.N. Chen (National Taiwan University, Taipei, Taiwan) is particularly noteworthy in connection with his studies on marine mollusks
and crustaceans. Sce Chen, S.N., Jong, K.J.; Kou, G.H. 1989. Cell cultures derived from tissues of penacid shrimp, Penaeus penicillatus, and hard clam,
Meretrix lusoria. In: Mitsuhashi, J.,ed. Invertebrate cell system applications. Vol. I]. Boca Raton, FL: CRC Press; p. 253-262. Sec also Professor Chen's
abstract, “Results of Tissue and Cell Culture of Fishand Aqualic Invertebrates,” in Appendix A to these proceedings. The abstract was submitted inresponse
to the invitation extended lo him to participate in the workshop.

Aneffort todevelop a coordinated international mullilaboratory program involving cell cultures of marine mollusks has already been implemented in France.
‘A comprehensive document that defines the objectives of the program and itemizes its activities, participating laboratories, and the personnel involved,
has been published. The document contains a wealth of bibliographic and other pertinent information pertaining to descriptive accounts of individual
projects and related activities with other laboratories in Europe, North America, and Asia. I thank Drs. A. Guillouzo and G. D’Orange for enlightening
me about this forward-looking, ambitious, and urgently needed program. Dr. D'Orange, coordinator of the program, may be contacted at: Laboratoire
de Biologic Marine, Université de Bretagne Occidentale, Faculté des Sciences, 29287 Brest.


Page 20

cholesterol and apolipoprotein in the up-and-down regula-
tion of gap junctions is becoming apparent (Meyer et al.
1990, 1991). Such activity is lipid concentration dependent
and involves complex molecular interactions at the mem-
brane level (Malewicz et al. 1990). This gap junction
research also confirms the interdependence of lipids and
proteins in cell culture systems.

CONCLUSIONS

Recent work with invertebrate cell culture systems
indicates cell surface (lipidic and extracellular matrix) speci-
ficities which significantly differ from a number of the
extant vertebrate cell culture system requirements (Goodwin
1988, 1989, 1991). At the same time, significant genetic
homologies are being revealed between vertebrate and in-
vertebrate bioactive peptides. These compounds have been
most useful in revealing the metabolic and functional con-
trols of normal as opposed to transformed vertebrate cells in
culture. In searching for new successful invertebrate cell
culturing systems, we should be guided by a careful com-
parison with results published on normal stem cells and
differentiation states rather than by transformed cell culture
requirements.

LITERATURE CITED

Allen, R.G. 1991. Oxygen-reactive species and antioxidant
responses during development: the metabolic paradox of
cellular differentiation. Proc. Soc. Exp. Biol. Med. 196:
117-129,

Blau, H.M.; Baltimore, D. 1991. Differentiation requires
continuous regulation. J. Cell Biol. 112: 781-783.

Bryant, P.J.; Schmidt, O. 1990. The genetic control of cell
proliferation in Drosophila imaginal discs. J. Cell Sci.
Suppl. 13: 169-189.

Cheesebeauf, M.; Padieu, P. 1984. Rat liver epithelial cell
cultures in serum-free medium: primary cultures and
derived cell lines expressing differentiated functions. In
Vitro 20: 780-795.

Chen, J.S.; Del Fa, A.; DiLuzio, A. 1976. Liposome-
induced morphological differentiation of murine neuro-
blastoma. Nature 263: 604-606.

Chiquet, M.; Masuda-Nakagawa, L.; Beck, K. 1988. At-
tachment to an endogenous laminin-like protein initiates
sprouting by leech neurons. J. Cell Biol. 107: 1189-1198.

Chitwood, D.J.; Lusby, W.R. 1991. Metabolism of plant
sterols by nematodes. Lipids 26: 619-627.

Devaney, E. 1985. Dirofilaria immitis: the moulting of the
infective larva in vitro. J. Helminthol. 59: 47-50.

Esko, J.D.; Nishijima, J.; Raetz,C.R.H. 1982. Animal cells
dependent on exogenous phosphatidylcholine for mem-
brane biogenesis. Proc. Nat. Acad. Sci. U.S.A. 79: 1698-
1702.

Fehon, R.G.; Johansen, K.; Rebay, I. 1991. Complex
cellular‘and subcellular regulation of notch expression
during embryonic and imaginal development of Dros-
ophila: implications for notch function. J. Cell Biol. 113:
657-669.

Franke, E.D.; Weinstein, P.P. 1983. Dipetalonema viteae
(Nematoda; Filarioidea): culture of third-stage larvae to
young adults in vitro. Science 221: 161-163.

Gelbart, W.M. 1991. A growth factor homologous gene
controlling pattern formation in Drosophila. In: Bellvé,
AR.; Vogel, H.J., eds. Molecular mechanisms in cellular
growth and differentiation. San Diego, CA: Academic
Press; p. 249-258.

Goodwin, R.H. 1988. The effect of lipids on the subculture
of differentiated cells from primary cultures of grasshop-
perembryonic tissues. In Vitro Cell. & Dev, Biol. 24: 388-
400.

Goodwin, R.H. 1989. Construction of peptoliposomes for
the incorporation of nutrient lipid supplements in insect
cell culture media. J. Tissue Cult. Meth. 12: 17-20.

Goodwin, RH. 1991. Replacement of vertebrate serum
with lipids and other factors in the culture of invertebrate
cells, tissues, parasites, and pathogens. In Vitro Cell. &
Dev. Biol. 27A: 470-478.

Hansen, E.L. 1976a. A cell line from embryos of
Biomphalaria glabrata (Pulmonata): establishment and
characteristics. In: Maramorosch, K., ed. Invertebrate
tissue culture research applications. New York: Aca-
demic Press; p. 75-99.

Hansen, E.L. 1976b. Application of tissue culture of a
pulmonate snail to culture of larval Schistosoma mansoni.
In: Kurstak, E.; Maramorosch, K., eds. Invertebrate tissue
culture: applications in medicine, biology and agriculture.
New York: Academic Press; p. 87-97.

Hansen, E.L. 1979. Initiating a cell line from embryos of the
snail Biomphalaria glabrata, Tissue Cult. Assoc. Man, 5:
1009-1014.

Hoffman, J.; Prochet, M., editors. 1984. Biosynthesis,
metabolism and mode of action of invertebrate hormones.
Berlin, Germany: Springer-Verlag; 519 p.

Hoffmann, F.M. 1989. Rolesof Drosophila proto-oncogenes
and growth factor homologs during development of the
fly. Curr. Top. Microbiol. Immunol. 147; 1-29.

Hoffmann, F.M. 1990. Developmental functions of
decapentaplegic, amember of the TGF-B family in Dros-
ophila. In: Mahowald, A.P., ed. Genetics of pattern
formation and growth control. New York: Wiley-Liss; p.
103-124.

Kaji, K.; Tanabe, H.; Nishi, N. 1991. Purification and
characterization of a growth factor produced by Parame-
cium tetraurelia. In Vitro Cell. & Dev. Biol. 27 (3, Pt.2):
48A.

Kano-Sueoka, T.; King, D.M.; Fisk, H.A. 1990. Binding of
epidermal growth factor to its receptor is affected by
membrane phospholipid environment. J. Cell Physiol.
145: 543-548.


While the majority of the known insect cell culture
nutritional requirements resemble those of vertebrate cells
(Mitsuhashi 1989), there are apparently substantial differ-
ences between vertebrate and invertebrate lipid require-
ments for cells in culture (Chitwood and Lusby 1991;
Goodwin 1991; Thompson et al. 1991). Some of the
apparent differences may actually be due to the differences
between transformed and nontransformed cells in culture,
however (Goodwin 1991). Various lipid complexes have
been formulated by researchers to supplement cultured
vertebrate cells either with or without vertebrate serum
supplementation (Chen et al. 1976, Esko et al. 1982;
Cheesebeauf and Padieu 1984). More recently, commer-
cially available lipid-hormone concentrates and complexes,
designed for vertebrate cells, have been used successfully
for the nutrient supplementation of some existing insect cell
lines (Goodwin 1991).

While my own research has involved liposome con-
struction for more specific lipid supplementations (Goodwin
1989), this has not solved problems in the development of
supplementations and a medium formulation that supports
the continuous culture of epithelial cell types from grasshop-
per embryos. Certain epithelial cells in grasshoppers make
up the major fat body, a liver-like tissue that is the sole
replication site for the grasshopper entomopox viruses (Good-
win 1988). Inthis andsome of my past lipid-related research
efforts, I noticed similarities to the problems around verte-
brate serum supplementations that were discussed earlier by
Eder Hansen and others dealing with noninsect invertebrate
cell cultures. Dr. Hansen described the extreme sensitivity
displayed by her molluscan cells (from Biomphalaria
glabrata embryos) to changes in vertebrate serum supple-
mentation evident when she changed serum lots or used
different serum sources (Hansen 1976a). It was only through
the careful testing of various vertebrate serum sources that
she was able eventually to isolate the only molluscan cell
line available to date (Hansen 1979). Since the lipid content
of animal serum is highly variable and serum lipid oxidation
generates toxic products, it may be that her cells were
responding to serum lipid variations. Also, her work with
schistosomes in her B. glabrata cell cultures also indicated
lipid oxidation problems, since snail hemolymph supple-
mentation was successful only when the hemolymph was
pretreated with (0.1 mM) dithiothreitol, a strong reducing
agent (Hansen 1976b). Likewise, the serum source was
considered to be the “most important factor” in the support
of molting and growth of filarial nematodes in vitro (Devaney
1985). And only one serum batch was effective in support-
ing the development of third-stage larval filarial nematodes
to adults in vitro (Franke and Weinstein 1983). Similar
serum problems led to the incorporation of a separate sterol
concentrate supplementation for the improvement of tick
cell cultures (Munderloh and Kurtti 1989). In my opinion,
these problems all strongly suggest the need for special
attention to probable specific cell-culture-essential lipid
requirements.

Another problem preventing the long-term or continu-

Page 19

ous subculturing of grasshopper embryonic epithelial cells
was my inappropriate dependence upon phosphatidylcho-
line-based phospholipids for epithelial cell culture (Goodwin
1989). It is now becoming evident that, in addition to the
powerful inhibitory influence of transforming growth fac-
tor- beta (TGF-B) in vertebrate sera (Goodwin 1991; Moses
et al. 1991), epithelial cells (and other cell types) are
sensitive to the ratio of various phospholipids present, and
in particular to the phosphatidylcholine-phosphatidyletha-
nolamine ratio (Kano-Sueoka et al. 1990; Sutton et al.
1991).

A more obvious impediment to the cell culture of
various invertebrate groups, as well as the culture of
nonfibroblastoid invertebrate cell types, is our lack of un-
derstanding at the cell level of hormone and growth factor
activity. The juvenile and molting hormones (ecdysteroids)
have been widely investigated in vivo, but less so in cell
culture. The ecdysteroids are comparable in some respects
to vertebrate steroid hormones, and seem to overlay the
network of more locally active peptide factors, lectins,
proteoglycans, and other extracellular matrix agents, pros-
taglandins, and various specialized lipidic molecules. While
a number of specific invertebrate hormones have been
characterized (Hoffman and Porchet 1984), invertebrate cell
growth factors are poorly known. It is most fascinating that
homologues of EGF (Thomas et al. 1991) and TGF-B
(Gelbart 1991) have been recognized in a number of inver-
tebrates extending down as far as the Protozoa (Kaji et al.
1991) and viruses (Goodwin 1991). These factors have been
compared to their vertebrate counterparts (Massague 1990),
and their interactions in imaginal discs have been explored
(Bryant and Schmidt 1990). It is heartening that the func-
tional roles of these growth-factor homologues also are
being revealed so quickly (Hoffmann 1989, 1990; Fehon et
al. 1991).

Other newly described cell-active factors include an
invertebrate laminin, active in the sprouting of neurons,
whichis not cross-reactive with vertebrate laminin (Chiquet
etal. 1988). This factor may be related to the nerve growth
factor/nerve regrowth activity described recently from a
snail (Ridgeway eral. 1991). Now that the axiom that nerve
growth does not occur in adult vertebrate animals has been
contradicted by extensive experimental evidence (Nottebohm
1985; Nottebohm et al. 1990), it seems more likely that
invertebrate cell culturesystems could help reveal the growth
control mechanisms for nerve cells in general.

The sequence of cell differentiation (i.e., neurite sprout-
ing) after growth and division may not be a fixed unidirec-
tional chain of events. Apparently, eukaryotic cellular
differentiation can be reversed and requires continuous
tegulation for maintenance (Blau and Baltimore 1991).
Since the cellular oxidation state is a major influence con-
trolling differentiation (Allen 1991), it follows that cellular
lipid composition and activation, and therefore the supply of
essential cellular lipids, will be critical tothe maintenance or
reversal of differentiation. Proof of such relationships may
be slow to surface, but already the reversible activity of


Page ii

NOTE ON SPECIES NAMES

The Northeast Fisheries Science Center's policy on the use of scientific names in technical publications and reports is
to follow the American Fisheries Society’s (AFS) lists of common and scientific names for fishes (Robins et al. 1991)*,
mollusks (Turgeon et al. 1988), and decapod crustaceans (Williams et al. 1989)*. This policy applies to all issues of the
NOAA Technical Memorandum NMFS-F/NEC series.

* Robins, C.R. (chair); Bailey, R.M.; Bond, C.E.; Brooker, J.R.; Lachner, E.A.; Lea, R.N.; Scott, W.B. 1991. Common and scientific names of fishes from
the United States and Canada. 5th ed. Amer. Fish. Soc, Spec. Publ. 20; 183 p.

> Turgeon, D.D. (chair); Bogan, A.E.; Coan, E.V.; Emerson, W.K.; Lyons, W.G.; Pratt, W.L.; Roper, C.F.E.; Scheltema, A.; Thompson, F.G.; Williams,
J.D. 1988. Common and scientific names of aquatic invertebrates from the United States and Canada: mollusks. Amer. Fish. Soc, Spec. Publ. 16; 277
Pp

© Williams, A.B. (chair); Abele, L.G.; Felder, D.L.; Hobbs, H.H., Jr.; Manning, R.B.; McLaughlin, P.A.; Pérez Farfante, I. 1989. Common and scientific
names of aquatic invertebrates from the United States and Canada: decapod crustaceans. Amer. Fish Soc. Spec. Publ. 17; 77 p.


Page 10

in mammalian fibroblasts and cells lining small blood
vessels. At 25 ng/ml, it promoted long-term venom
gland cell survival.

Insulin: Used as an effective replacement for fetal
bovine serum (FBS) in a medium for Drosophila cell
lines, and insulin-like hormones have been detected in
insect hemolymph.

Cyclic adenosine monophosphate (cAMP): This
stimulates terminal differentiation in many cells and it
maintained venom gland cell viability. In the absence
of cAMP, the cells developed granular cytoplasm,
lysed, and degenerated within 4-5 mo. Those kept in
cAMP-supplemented media remained viable for up to
12 mo. Cultures maintained in 0.05M cAMP for 3 mo
and then switched to cAMP-free medium degenerated
within 1 mo.

Isoproterenol: A catecholamine that regulates intra-
cellular levels of cAMP and Ca++ from the cell surface.
Since cAMP was beneficial, this compound was used to
elevate intracellular levels of cAMP. At 5x10°M, it
promoted formation of hollow multicellular vesicles.
Other materials known to elevate cAMP levels (cholera
toxin, epinephrine, etc.) were not effective.

Cyclic guanosine monophosphate: At 10°M, this
produced a very striking stimulation of cell activity
(initial migration and multiplication) that was more
pronounced when combinedwith cAMP.

Buffers: Cell culture media did not have enough
buffering capacity and pH dropped from the initial
value of 7.0 to 6.2-6.0 in 4 days. We evaluated numer-
ous buffers at several concentrations: BES [N,N-bis(2-
hydroxyethyl)-2-aminoethanesulfonic acid]; MES (2-
(N-morpholino)-ethanesulfonic acid); ACES [N-2-
acetamido-2-aminoethanesulfonic acid]; and HEPES
{N-2-hydroxyethylpiperazine-N'!-2-ethanesulfonic
acid]. The 0.1M BES kept the pH constant and cells in
the best morphological condition.

PRIMARY CULTURES

Embryonic tissues were used to initiate flea cell lines as
they are often the tissue of choice because cells are actively
multiplying and numerous invertebrate and vertebrate lines
developed from embryos.

The eggs were disinfected by immersion in 1% NaOCl+
0.1% Tween 80 for 2 min. The wetting agent in the NaOCl
is important as it permits the entire surface of eggs or
complete insects to be wetted, thus ensuring that all cuticular
depressions are exposed to the disinfectant. Eggs are then

rinsed four times in sterile water and dried on sterile blotting
Paper.

Since the amount of tissue from eggs is small and there
is some suggestion that tissue-medium ratio is an important
consideration, we cultured the tissues in standing drops. A
volume of 0.15 ml was placed in the middle of 35x10-mm
Lux petri dishes, the rinsed eggs transferred to the drop (10
per drop), and the embryos pulled apart with tweezers. We
often refeed the cultures after 24 hr as this promotes cell
viability. After the first refeeding, we replace mediumevery
7-10 days. However, the best schedule for medium replace-
ment should be experimentally determined. Refeeding is
done by using Pasteur pipets drawn out toa fine point; and
medium is drawn up the perimeter of the drop. This prevents
tissue from being aspirated into the pipet. Fresh medium
(0.1 ml) is added immediately to the cultures and they are
incubated at 100% relative humidity to prevent desiccation.
We have kept cells alive for up to 12 mo with this technique.

If attached cells are multiplying, at some point, the
surface area under the drop must be increased to give room
for more cells. When about 60-75% of the area under the
drop has cells, the entire surface is covered by adding 2.0 ml
medium. The culture may be “reseeded” by gently flushing
medium over the attached cells which frees some of them.
They attach to other areas of the dish and form colonies.

Itis important to keep comparatively large fragments of
embryos in the drops because the presence of 4-6 floating
pieces promoted the multiplication of attached cells. Of 48
cultures, only those (4) where tissue fragments were present
developed into cell lines, and, without fragments, cells did
not become dense enough to subculture.

Another reason for maintaining suspended tissue frag-
ments is that they can be a source of continuously growing
cells, With embryos of the codling moth, cells did not attach.
After 1 moin culture, cells began to migrate from the interior
of the suspended tissues, and many layers formed on the
exterior surface of the fragments. These cells descended to
the bottom of the flask and formed colonies of attached cells.
Within 2 mo, the primary culture contained many suspended
cells and a monolayer that was subcultured.

If tissues are to be dissociated with trypsin, we find that
incubation in 0.25% trypsin for 12 hr at 5°C causes less
damage than incubating at 28°C for 20-30 min. After trypsin
treatment, it is pipeted off and soybean trypsin inhibitor
(0.1%) added directly to the tissue. The pieces are then
transferred to standing drops of media, pulled apart, and
agitated with tweezers. The use of soybean trypsin inhibi-
tor, which combines with trypsin and ties up the active sites,
reduces cell damage in comparison to that observed when
trypsin was rinsed off.

SUBCULTURING

A problem frequently encountered is that cells die after
subculturing even though those in the primary culture are


(continued from inside front cover)

79. Contaminants in Sediment and Fish Tissue from Estuarine and Coastal Sites of the Northeastern United
States: Data Summary for the Baseline Phase of the National Status and Trends Program Benthic
Surveillance Project, 1984-1986. By Vincent S. Zdanowicz and Donaid F, Gadbois. December 1990. x + 138p.,
67 figs., 21 tables. NTIS Access. No. PB92-147453/AS.

80. Distribution of Sexually Immature Components of 10 Northwest Atlantic Groundfish Species Based on
Northeast Fisheries Center Bottom Trawl Surveys, 1968-86. By Susan E. Wigley and Wendy L. Gabriel. January
1991. iv + 17 p., 23 figs., 3 tables. NTIS Access. No. PB92-101617.

81. Status of the Fishery Resources Off the Northeastern United States for 1990. By Conservation and
Utilization Division, Northeast Fisheries Center. January 1991. iv + 130 p., 50 figs., 82 tables. NTIS Access. No.
PB91-213785. .

82. Response of the Habitat and Biota of the Inner New York Bight to Abatement of Sewage Sludge Dumping:
Third Annual Progress Report—1989. By Anne L. Studholme, Merton C. Ingham, and Anthony Pacheco, eds.
February 1991. vi + 57 p., 74 figs., 20 tables, 1 app. NTIS Access. No. PB91-208199.

83. Organic Contaminants in Hepatic Tissues of Lobster and Flounder at the New York Bight “12-Mile”
Sewage Sludge Dumpsite: 1987-88. By A.F.J. Draxler, Paul Hauge, and Ashok D. Deshpande. July 1991. iii +
10 p., 3 figs., 8 tables. NTIS Access. No. PB93-114635/AS.

84. Trophodynamics of Select Demersal Fishes in the New York Bight. By Frank W. Steimle and Russell
Terranova. July 1991. iv + 11 p., 1 fig., 16 tables. NTIS Access. No. PB92-157999/AS.

85. Factors Influencing Spring Distribution, Availability, and Recreational Catch of Atlantic Mackerel
(Scomber scombrus) in the Middle Atlantic and Southern New England Regions. By William J. Overholtz, Reed
S. Armstrong, David G. Mountain, and Mark Terceiro. August 1991. iii + 13 p., 9 figs., 3 tables. NTIS Access. No.
PB92-160209.

86. Status of Fishery Resources off the Northeastern United States for 1991. By Conservation & Utilization
Division, Northeast Fisheries Science Center. September 1991. iii + 132 p., 55 figs., 72 tables. NTIS Access. No.
PB92-113786.

87. Evidence of Structural Change in Preferences for Seafood. By Steven F. Edwards. January 1992. iii +12
p., 3 figs., 1 table. NTIS Access. No. PB93-114650/AS.

88. Synopsis of Principal Diseases of the Blue Crab, Callinectes sapidus. By Gretchen A. Messick and Carl J.
Sindermann. January 1992. iii + 24 p., 13 figs., 2 tables. NTIS Access. No. PB92-219757.

89. Proceedings of the NEFC/ASMFC Summer Flounder, Paralichthys dentatus, Aging Workshop, 11-13
June 1990, Northeast Fisheries Center, Woods Hole, Mass. By Frank P. Almeida, Raoul E. Castaneda, Roman.
Jesien, Richard E. Greenfield, and John M. Burnett. January 1992. iii + 7 p., 8 figs., 2 tables. NTIS Access. No.
PB93-114643/AS. .

90. Fish and Megainvertebrates Collected in the New York Bight Apex during the 12-Mile Dumpsite Recovery
Study, July 1986-September 1989. By Stuart J. Wilk, Robert A. Pikanowski, Anthony L. Pacheco, Donald G.
McMillan, Beth A.-Phelan, and Linda L. Stehlik. October 1992. iv + 78p., 9 figs., 2 tables, 2 app. NTIS Access.
No. PB93-158772.

91. The Large Marine Ecosystem (LME) Concept and Its Application to Regional Marine Resource
Management - - 1-6 October 1990, Monaco: Conference Summary and Recommendations. By Kenneth Sherman
and Thomas L. Laughlin, eds. October 1992. v+37p., 3 app. NTIS Access. No. PB93-185965.

92. Report of the Meeting of the ad hoc Committee on Large Marine Ecosystems, 22-23 March 1991, UNESCO
Headquarters, Paris, France. By Kenneth Sherman and Thomas L. Laughlin, eds. October 1992. iii+ 19p.,1
fig., 4 app.


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Page 16

CULTURED LOBSTER OLFACTORY NEURONS:
UTILITY FOR STUDYING SIGNAL TRANSDUCTION®

D.A. Fadool, W.C. Michel’, and B.W. Ache
Whitney Laboratory and Departments of Zoology and Neuroscience
University of Florida
St. Augustine, Florida 32086

We attempted to sustain crustacean olfactory receptor
neurons in primary cell culture to provide a system for
studying olfactory transduction. Olfactory receptor cells in
crustaceans, as in many animals, are long, thin, bipolar
neurons that are not amenable for voltage-clamping odor-
activated currents. Here we report a protocol that allows
olfactory receptor neurons harvested from Panulirus argus,
the Caribbean spiny lobster, to survive in primary culture for
upto 23 days. The cultured cells are more compact than their
counterparts in vivo, yet display voltage- and odor-activated
properties similar to those of lobster olfactory receptor cells
in situ.

Cell cultures were prepared from the distal third of the
lateral antennular filament (olfactory organ) of intermolt
lobsters, which had previously been briefly rinsed in 10%
Listerine in Panulirus saline. Clusters of the olfactory
receptor cell somata were isolated, proteolytically treated
with 0.25 mg/ml of L-cysteine (1.25 mg/ml)-activated pa-
pain for 50 min, and then mechanically dissociated and
plated at a density of 12x10‘ cells/ml (per 2-cm? well) with
a61% yield. Neurons were cultured in a modified Liebowitz
L15 media supplemented with salts to mimic Panulirus
saline, basic minimal essential vitamins, L-glutamine, low
dextrose, and either 6% FCS or 10% lobster hemolymph.
Only one-half of the media was changed every fourth day to
allow accumulation of any required neurotrophic factor.

Neurite outgrowth and cell viability were strongly
affected by the type of substrate, the presence and timing of
serum supplementation, and the length of time the animals
were held in captivity. The cells sprouted processes within
2 hr after plating and showed optimal survival when plated
on poly-D-lysine hydrobromide-coated (molecular weight
49,300-53,000) glass coverslips (2.5-5.0 g/cm?) or when
grown on hemolymph clots. The cells did not adhere to
untreated glass coverslips or collagen or laminin substrates,
and adhered--but with little neurite outgrowth--to commer-
cial plastic tissue culture wells or dishes (Coming and
Falcon #1008, respectively). The cells did not adhere
uniformly to the substrate when FCS was provided immedi-
ately upon plating (vs. 2-12 hr later), nor when the cells were
kept under room light or continually agitated. The cells
survived best when maintained at saturation humidity in a
modular incubator chamber (Billups-Rothenberg) inside a
low-range temperature incubator at 24°C (Hotpack). The

© Supported by Office of Naval Research grant NOO14-90-J-1566.

cells survived equally well between 965 and 1082 mOsm,
even though the actual hemolymph osmolarity is closer to
the upper limit of this range -- 1079+37.5 mOsm (average +
standard error of the mean; N = 3). The addition or omission
of HEPES or nerve growth factor (NGF-7s) did not notice-
ably affect either longevity or neurite outgrowth. Plating
density dropped by one-third within the first hours of
plating, with few cells extending processes, when tissue was
harvested from animals held 8 wk or longer, compared to
that from animals held no more than 3 wk. Preconditioned
media derived from the target organ (brain and olfactory
neurophil), collected at 24- and 60-hr intervals, had no
obvious effect on either longevity or neurite outgrowth
when used to supplement up to 50% of the culture medium.

Five morphologically distinct types of “neuron-like”cells
could be defined based on their number and type of process.
The somata ranged from 8 to 16 ym in diameter. Processes,
when present, were less than 160-ym long. Cells of all five
types were electrically excitable (N of all types = 50);
voltage elicited inward and outward macroscopic currents
that activated concurrently at approximately -30 mV. Step-
ping the cells in increments of 10mV from -60 (rest) to +30
mV activated a maximum transient inward current of
332.3£27.9 pA. The same protocol activated a maximum
sustained outward current of 756.9+30.0 pA. The cultured
olfactory neurons displayed odor-activated properties that
were not significantly different from those of receptor cells
in situ. Odors evoked currents of opposite polarity, and
could do so in the same cell. Odor-evoked currents of both
polarities were associated with a decrease in membrane
resistance and a corresponding decrease in the charging time
of the membrane. The current magnitudes of both polarities
were dose-dependent, with thresholds as low as 10° M, the
lowest concentration tested. The magnitude of the inward
current (39.2+3.0 pA, N = 56) was significantly greater than
that of the outward current (20.1+1.4 pA, N = 49; t' statistic,
ps0.05). A sample population of cells (N = 53) presented
sequentially with the same five odors varied in their degree
of tuning. Some cells were narrowly tuned, responding to
only one or two of the test odors, while other cells were
broadly tuned, responding to up to four test odors. No cell
responded to the entire complement of test odors.

The latency to the onset of the outward and inward
current was consistent with second-messenger mediation

1 Current address: Department of Physiology, University of Utah School of Medicine, 410 Chipeta Way, Salt Lake City, UT 84108.




NEOPLASIA IN MARINE
INVERTEBRATES

High prevalences of tumors and related lesions, both
internal and external, have been observed in marine animals,
including invertebrates inhabiting the coastal ecosystems of
North America. Again, state and Federal agencies, conser-
vationists, the fishing industry, and the public are very
concerned about these findings, particularly from the view-
points of human health, edibility of resources, and safety, as
well as from the perspective of maintaining environmental
(habitat) integrity. A number of investigators have under-
taken activities to develop “immortal” cell culture systems
employing what appears to be neoplastic cells of mollusks
(oysters and clams) as found in some feral populations.
Others, in order to study and compare neoplasm initiation
and promotion mechanisms among the invertebrates and
higher forms, are using exposure of intact animals, tissues,
or cells to various carcinogens or mutagens. Some recent
studies also focus on the identification, characterization, and
activities of exogenous and endogenous growth promoter
particles (viruses), as well as segments of nucleic acids
(genes or their precursors -- oncogenes) recognized from
tumorous invertebrate tissues. Attempts are also being
made to determine and compare the nature of these elements
with homologous entities as found in “lower” or “higher”
taxa, including humans. Although some progress has been
made in understanding oncogenetic processes through these
studies, development of immortal cell lines using neoplastic
cells and isolates from marine invertebrates has not yet been
reported; efforts along these lines are, however, continuing.

MARINE BIOTECHNOLOGY

Within recent years, there has been a veritable explo-
sion of human activity in the field of biotechnology. Appli-
cation of the term biotechnology to a number of activities
involving human intrusions into and rearrangements of
biological conformations and processes does not mean
necessarily a new field or discipline has been developed or
that a new word has been coined. Manipulations of living
organisms (particularly microbial or cellular forms) and
their processes so that the outcome serves the needs of
mankind have been practiced since the dawn of civilization,
even before. The use of plant and animal (including micro-
bial agents) enzyme systems, extracts, and parts to accom-
plish various chemical and structural conversions of organic
materials is well known, as are the traditional methods of
plant and animal selection for breeding and hybridization. It
was advances in the knowledge and understanding of gene
actions-interactions and the development of DNA manipu-
lation technology that were and continue to be major ele-
ments responsible for progress in biotechnology or genetic
engineering. Application of these biotechnological ap-
proaches allows for the creative design and control of

Page 3

biological-genetic components and processes that can bring
about structural changes or alter cellular events that, in turn,
influence metabolism, reproduction, growth, development,
and behavioral characteristics (i.e., phenotype).

Considerable attention has been given to the use of in
vitro cell, tissue, and organ cultures of marine plants and
fishes for biotechnological applications. On the other hand,
only limited activity has been directed toward use of marine
invertebrate cell, tissue, and organ culture for biotechno-
logical-genetic engineering purposes--largely because long-
term, in vitro, marine invertebrate cell cultures for all
intents and purposes do not exist or are unavailable.

As expressed in the preface of these proceedings, the
needs and potential uses of long-term marine invertebrate
cell culture systems are manifold. We will hear about some
of the efforts and progress being made in marine biotechno-
logical applications during this workshop; however, it is
apparent that much more could and should be done by the
many marine laboratories throughout the world to achieve
competency for culturing cells from the several phyla of
organisms inhabiting the sea and estuaries. The possibilities
for biotechnological breakthroughs to develop new prod-
ucts and new genetic assemblages are limitless.

REFERENCES

Marine Mollusks

Bevelander, G.; Martin, G. 1949. Culture of mantle tissue
of marine molluscs. Anat. Rec. 105: 614.

Brewster, F.; Nicholson, B.L. 1979. Jn vitro maintenance
of amoebocytes of the American oyster (Crassostrea
virginica). J. Fish. Res. Board Can. 36: 461-470.

Cecil, J.T. 1969. Mitoses in cell cultures from cardiac
tissues of the surf clam Spisula solidissima. J. Invertebr.
Pathol. 14: 407-412.

Ellis, L.L.; Bishop, S.H. 1989. Isolation of cell lines with
limited growth potential from marine bivalves. In:
Mitsuhashi, J.,ed. Invertebrate cell system applications.
Vol. II. Boca Raton, FL: CRC Press; p. 243-251.

Ellis, L.L.; Brodey, M.M.; Bishop, S.H. 1985. Preparation
of cells from marine molluscs for culture. In Kurstak, E.,
ed. Techniques in life sciences. Vol. C1 (Part C107).
Setting up and maintenance of tissue and cell cultures.
Limerick, Ireland: Elsevier Scientific Publishers; p. 1-15.

Hetrick, F.M.; Stephens, E., Lomax, N.; Lutrell, K. 1981.
Attempts to develop a marine molluscan cell line. Mary-
land Sea Grant Program Publication No. UM-SG-TS-81-
06. Available from: University of Maryland, College
Park, MD.

Li, M-F.; Stewart, J.E.; Drinnan, R.E. 1966. In vitro
cultivation of oyster cells in the oyster, Crassostrea
virginica. J. Fish. Res. Board Can. 23: 595-605.

Perkins, F.O.; Menzel, R.W. 1964. Maintenance of oyster
cells in vitro. Nature 204: 1106-1108.


Page 5

MODELS TO ILLUSTRATE THE NEEDS, USES, AND BENEFITS
OF MARINE INVERTEBRATE CELL CULTURE

Carol L. Reinisch
Department of Comparative Medicine
Tufts University School of Veterinary Medicine
North Grafton, Massachusetts 01536-1895

Marine invertebrates offer excellent models with which
to study problems in malignant transformation and cell
activation. In the accompanying talk, I will present two
models--one in a marine mollusk, and the second in an
echinoderm--and use these examples to illustrate the state of
the field and how the culture of cells could aid greatly in the
resolution of fundamental issues in cell biology.

Mya arenaria, the softshell, develops a leukemia. The
leukemia cells are detected first in the hemolymph and then
in solid tissue. Monoclonal antibodies generated to the
leukemia cells recognize a 190-kD, cell-surface protein.
The structure, sequence, and function of this protein, which
is not detected on normal cells, are totally unknown.

In fact, we do not know if leukemia cells are trans-
formed normal hemocytes; an experiment cannot be done
directly until the amoebocytes are cultured in vitro. Rather,
our approach has been (and will continue to be) the use of
immunologic and molecular probes. For example, we will
generate acDNA library for both types of cells. By subtrac-
tive hybridization, we hope to ascertain which sequences are
unique to both cell types.

Inasecond system, developed by R.A. Prendergast and
his collaborators at Johns Hopkins University, coelomocytes
from Asterias forbesi have been shown to contain a 39-kD
protein. called sea star factor (SSF). This protein has
profound activity on mammalian cells. SSF prevents the
division of normal (unprimed) T lymphocytes to antigenic
stimulation, and inhibits antibody responses by limiting the
availability of lymphokines produced by helper T cells.
However, in the invertebrate, when used in vivo in ELVAX
capsules, SSF is chemotactic, drawing amoebocytes to the
chamber within 24-48 hr.

Inthe echinoderm model, cell culture would permit the
study of cytokines and growth factor receptors. Using
Prendergast’s approach, the phylogeny of growth factors
and their receptors could be studied--work which right now
is relegated to in vivo research because no adequate cell
culture system exists.

The list of examples is endless, and additional areas will
be covered during my presentation. Concerning initiatives,
I firmly believe that a high priority in this field should be the
establishment of tissue culture facilities solely devoted to
the technology required to culture marine invertebrate cells.
Funding should be cooperative between agencies commit-
ted to work in the field and must be long term.

In summary, immunologists are investigating clams
and sea stars to resolve fundamental questions in cell biol-
ogy. As examples, we are studying adhesion, transforma-
tion, and cell activation. The advent of cell culture will
greatly facilitate our research and that of many others in this
explosive field of work.

REFERENCES
Mollusks

Miosky, D.; Smolowitz, R.; Reinisch, C.L. 1989. Leukemia
cell specific protein of the bivalve mollusc Mya arenaria.
J. Invertebr. Pathol. 53: 32.

Smolowitz, R.; Reinisch, C.L. 1986. Indirect peroxidase
staining using monoclonal antibodies specific for Mya
arenaria neoplastic cells. J. Invertebr. Pathol. 48: 139.

Smolowitz, R.; Miosky, D.; Reinisch, C.L. 1989. The
ontogeny of leukemic cells of the soft shell clam. J.
Invertebr. Pathol. 53: 41.

Echinoderms

Kerlin, R.L.; Cebra, J.J.; Weinstein, P.D.; Prendergast, R.A.
In press. Sea star factor blocks development of T-
dependent antibody secreting clones by preventing lym-
phokine secretion. Cell. Immunol.

Prendergast, R.A.; Lutty, G.A.; Scott, A.L. 1983. Directed
inflammation: the phylogeny of lymphokines. Dev. Comp.
Immunol. 7: 629.


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available from the original submission.




Page 14

emphasis of this workshop. The questions and answers are
to serve as a (sometimes provocative) basis of debate and
discussion.

1,

Question: What are the qualities desired in the target
species and life-cycle stage selected as the source of
primary tissues?

Answer: Species should be subject to propagation inthe
laboratory, ensuring yields of good quantities of mate-
tial, at any life-cycle stage (preferably pathogen-free).
Early developmental stages (embryos/larvae) are obvi-
ous subjects that have been exploited for this purpose.
If hematopoietic, gonad, or other proliferating tissues
are known to be available in juvenile or adult stages,
these may yield suitable material for primary cultures.

Question: How completely should primary tissues be
“digested” with the aim of obtaining single-cell suspen-
sions for primary culture?

Answer: Incompletely, as cells need the “nurturing”
environment provided by tissue fragments (v. “vesicles”
of insect cell culturists). A caveat is that fragments can
complicate the visual evaluation of culture quality.
Hansen sieved embryos to obtain single cells and small
aggregates; she liberated cells using trypsin-EDTA.

Question: Does the marine source of tissues targeted
here impose identifiable problems?

Answer: Unknown; however, problems establishing
lines from nonarthropod invertebrates have not been
restricted to marine species. The necessary high os-
motic values provide the scope for a great variety of
media additives and relatively high concentrations.

Question: If a mechanical process is used to achieve
tissue/embryo disruption, are precautions needed to
reduce the risk of damage from reactive oxygen species,
lysosomal proteases, and other potentially destructive
products?

Answer: Unknown; however, can this be achieved with
superoxide dismutase, catalase, and protease inhibitor
cocktails; low temperature?

Question: In the absence of knowledge about the
requirements for, and the chemical nature of, growth
factors in target species, can their necessity be assumed
and can their functions be met by nonspecific strategies
like bypassing cell-surface receptor-ligand binding,

ie., directly target second-messenger systems (“shot-
gun”) using activators of protein kinases, calcium iono-
phore, etc.?

Answer: This approach, using phorbol myristic acetate
and A23 187, has been used successfully to immortalize
fish lymphoid cells (Miller and Clem, pers. comm.‘).

Question: Whenadding precious, heterologous growth
factors to primary cultures, are there benefits to be
gained from: (1) enzyme pretreating the cells?; (2)
doing this in protein-free media?; and (3) allowing time
for regeneration of cell surface receptors?

Answer: Hansen trypsinized primaries “to advantage.”
Insect primaries may be released from inhibition by
trypsinization (Schneider, pers. comm.). Serum-free
media are less likely to bind up growth factors.

Question: Are suprapharmacological doses of hor-
mones, efc., justified in light of the evolutionary dis-
tances which separate the producing species from the
target species?

Answer: Yes.

Question: Is there a role for feeder layers?

Answer: Moot, in the absence of existing, osmotically
compatible cell lines. (This also precludes cell fusion
as an approach to be used with marine invertebrates.)
Question: When should primaries be used for terminal
(fatal) assays such as nucleic acid or protein synthesis,

or for live:dead determinations?

Answer: Only when success has been achieved by
maintaining primaries for long periods (months?).

. Question: Should nonadherent cells and aggregates be

included in efforts to propagate cell lines?

Answer: Yes. These may yield the cell line desired, and
can later be selected or encouraged to adopt an adherent

phenotype.

Other factors to consider are:

What are the barriers? Given that numerous investiga-
tors have successfully established primary cultures
from marine mollusks, much of what is written above
may seem redundant. Perhaps the questions and poten-
tial solutions we need to address should center on the

‘ Miller, M.W.; Clem, W.L. Department of Microbiology, University of Mississippi Medical Center, 2500 North State St., Jacksonville, MS 39216.

5 Schneider, I. Department of Entomology, Walter Reed Amy Institute of Research, Washington, DC 20307.


Recent issues in this series

64. The Shell Disease Syndrome in Marine Crustaceans. By Carl J. Sindermann, February 1989. v + 43 p., 5 figs.,
2 tables. NTIS Access. No. PB89-162523/AS.

65. Stock Assessment Information for Pollock, Pollachius virens (L.),in the Scotian Shelf, Georges Bank, and Gulf
of Maine Regions. By Ralph K. Mayo, Stephen H. Clark, and M. Christina Annand. April 1989. vi + 14 p., 6 figs.,
14-tables. NTIS Access. No. PB90-120676/AS.

66. Guidelines for Estimating Lengths at Age for 18 Northwest Atlantic Finfish and Shellfish Species. By Judith
A. Pentilla, Gary A. Nelson, and John M. Burnett, III. May 1989. iii + 39 p., 18 figs., 19 tables. NTIS Access. No. PB90-
120675/AS.

67. Response of the Habitat and Biota of the Inner New York Bight to Abatement of Sewage Sludge Dumping.
Second Annual Progress Report ~ 1988. By Environmental Processes Division, Northeast Fisheries Center. July
1989. vii + 47 p., 39 figs., 11 tables, 3 app. NTIS Access. No. PB90-125444/AS.

68. MARMAP Surveys of the Continental Shelf from Cape Hatteras, North Carolina, to Cape Sable, Nova Scotia
(1984-87). Atlas No. 3. Summary of Operations. By John D. Sibunka and Myron J. Silverman. July 1989. iv + 197
P., 36 figs., 2 tables. NTIS Access. No. PB90-160656/AS.

69. The 1988 Experimental Whiting Fishery: A NMFS/Industry Cooperative Program. By Frank P. Almeida,
Thurston S. Burns, and Sukwoo Chang. August 1989. v + 16 p., 9 figs., 11 tables, 1 app. NTIS Access. No. PB90-
160664/AS.

70. Summer Distribution of Regulated Species on Georges Bank with Reference to the 1988 Experimental
Whiting Fishery. By Frank P. Almeida, Sukwoo Chang, and Thurston S. Burns. September 1989. v+ 25 p., 74 figs.,
1 table. NTIS Access. No. PB90-206525/AS.

71. Allocation of Statewide-Reported MRFSS Catch and Landings Statistics between Areas: Application to
Winter Flounder. By Frank P. Almeida. September 1989. v + 18p., Sfigs., 6 tables, 2 app. NTIS Access. No. PB90-
246745.

72. Status of the Fishery Resources off the Northeastern United States for 1989. By Conservation and Utilization
Division, Northeast Fisheries Center. December 1989. iv + 110p., 50 figs., 93 tables. NTIS Access. No. PB90-170622/
AS.

73. Surface and Bottom Temperature Distribution for the Northeast Continental Shelf. By David G. Mountain
and Tamara J. Holzwarth. December 1989. iii + 32 p., 31 figs. NTIS Access. No. PB90-252172/AS.

74. Shell Disease of Crustaceans in the New York Bight. By C.J. Sindermann (chair.), F. Csulak (EPA coord.), T.K.
Sawyer (rapp.), R.A. Bullis, D.W. Engel, B.T. Estrella, E.J. Noga, J.B. Pearce, J.C. Rugg, R. Runyon, J.A. Tiedemann,
and R.R. Young. December 1989. vii + 47 p., 7 figs., 4 tables, 4 app. NTIS Access. No. PB90-195058/AS.

75. An Indexed Bibliography of Northeast Fisheries Center Publications and Reports for 1988. By Jon A. Gibson.
December 1989. iii + 32 p. NTIS Access. No. PB91-112912.

76. Finfish Maturity Sampling and Classification Schemes Used during Northeast Fisheries Center Bottom
Trawl Surveys, 1963-89. By Jay Bumett, Loretta O'Brien, Ralph K. Mayo, Jeffrey A. Darde, and Margot L. Bohan.
December 1989. iii+14 p., 11 tables, 1 app. NTIS Access. No. PB91-128389/AS.

77. Shell Disease among Red Crabs Inhabiting Submarine Canyons of the New York Bight. By Randall R. Young.
December 1989. iii+9 p., 18 figs., 5 tables. NTIS Access. No. PB90-194762/AS.

78. Seasonal Distribution Patterns of Commercial Landings of 45 Species off the Northeastern United States
during 1977-88. By Sukwoo Chang. October 1990. v+130 p., 246 figs. NTIS Access. No. PB91-160846.

(continued on inside back cover)


Page 22

induce the formation of reactive oxygen intermediates (ROI),
and release proteolytic and other enzymes capable of harm-
ing cells. Precautions can include not only protease inhibi-
tors (quite widely used), but also scavengers (such as
superoxide dismutase and catalase), for ROI.

The question was raised about the possible usefulness
of attempts to trigger second-messenger systems directly
that are normally triggered by receptor-ligand interactions
with growth factors. For example, phorbol esters and
calcium ionophores might be so used. The report also
appealed for retention in cultures of fragments, nonadherent
cells, and anything else alive even when the morphological
characteristics of such items are not those desired in a cell
line; anything that grows should be accepted, and character-

istics such as adherent phenotype can be manipulated or
selected at a later time.

Finally, it should be noted that, in general, we do not
even know where in the cell cycle that blocks to propagation
arise in marine invertebrate cells in vitro. Are blocks to
propagation inadvertently introduced in preculture proto-
cols? Perhaps more fundamentally, we seldom, if ever,
know what the putative blocks are. Could they be: (1) lack
of essential growth factors?; (2) occupation of receptors by
nonstimulatory analogues of growth factors?; (3) lack of
essential nutrients?, and/or (4) presence of growth inhibi-
tors? Systems need to be developed to rule in or out
systematically each of these and other potential blocks to
cell propagation.


(81.0+10.6 and 186.2+31.3 msec, respectively), so we in-
vestigated this possibility. Pharmacological experiments
implicated guanosine triphosphate (GTP)-binding proteins
(G-proteins) in the coupling of cell-surface receptors to the
intracellular effectors that produce the currents of both
polarities. Including GTPyS (a nonhydrolyzable analog of
GTP) in the patch pipette increased odor-evoked currents of
both polarities over control (same cell without analog) by
244.2490.0% in 8 of 10 cells tested. Including GDPBS (a
nonhydrolyzable analog of guanosine diphosphate) in the
Pipette decreased odor-evoked currents of both polarities
(N= 9) by 50.0£10.0%. Thirty-hour incubation with 1-ym
pertussis toxin or 50-m cholera toxin had no significant
effect on the odor response, suggesting that the G-proteins
associated with lobster olfactory transduction are bacterial-
toxin insensitive.

Pharmacological experiments implicated a phospho-
lipid second messenger in the excitatory transduction cas-
cade. When 2.4x10°M inositol 1,4,5 triphosphate (IP,) was
introduced into the cultured cell through the patch pipette, 5
of 16 cells produced a sustained inward current (range of 20-
73 pA) froma holding potential of -60 mV. In four cells that
could be double patched, first creating a seal without TP, in
the pipette (control) and then creating a second seal with IP,
included (test), IP, increased the odor-evoked inward cur-
rent 336.2+113.2% over that of the contro]. Ruthenium red
(10 uM), an IP,-regulated Ca** channel blocker, attenuated
the IP,-induced increase in the odor-evoked current in three
out of the four cells.

These types of findings support our initial contention
that culturing the olfactory receptor neurons should allow us

Page 17

to investigate transduction-related questions that are techni-
cally difficult to address in the cells in situ. For example,
concern that membrane impermeant probes (e.g., those
targeted against IP,) that, in situ, must be introduced through
the patch pipette into the soma may not diffuse to their
suspected site of action in the outer dendrites, is minimized
in the morphologically compact cultured cells. The cultured
cells that lack processes, but still respond to odors, presum-
ably insert the appropriate channels into the soma mem-
brane, which can be readily patched. Indeed, work in
progress indicates this to be the case. The cultured system,
therefore, appears to be a fruitful model for studying signal
transduction in olfaction.

REFERENCES

Ache, B.W.; Derby, C.D. 1985. Functional organization of
olfaction in crustaceans. Trends Neurosci. 8(8): 356-360.

Fadool, D.A.; Ache, B.W. 1992. Single channel and
immunocytochemical evidence for inositol 1,4,5-triphos-
phate as a second messenger in lobster olfactory neurons.
Chem. Senses 17:621.

Fadool, D.A.; Michel, W.C.,; Ache, B.W. 1991. G-proteins
and inositol-phospholipid metabolism implicated in odor
response of cultured lobster olfactory neurons. Chem.
Senses 16: 518-519.

Fadool, D.A.; Michel, W.C.; Ache, B.W. 1991. Sustained
primary culture of lobster (Panulirus argus) olfactory
receptor neurons. Tissue & Cell 23: 719-732.

MARINE SPONGE CELL CULTURE

S.A. Pomponi, R. Willoughby, and J.E. Armstrong
Division of Biomedical Marine Research
Harbor Branch Oceanographic Institution
Ft. Pierce, Florida 34946

More marine natural products have been derived from
sponges than any other invertebrate group. Research on
compounds with potential therapeutic application often
requires a greater amount than can be supplied by collection
of the organisms. The objective of our research is to develop
sponge cell culture methods as an alternative to large-scale
collections for bulk production of potential therapeutic
agents derived from marine natural products.

A locally available sponge was selected as a model
system, and a procedure was developed for initial dissocia-

tion of tissue, attachment of cells, and control of contamina-
tion. Primary cultures are initiated from both adult and
larval sponges. The adult culture inoculum consists of a
monodisperse suspension. The larval cultures may be
started from similarly dispersed larval cells or from intact
larvae. Both types of cultures may produce functional
sponges.

The cultures are not axenic, and quantification of at-
tachment and growth has been difficult. The presence of
contaminating cells and the heterogeneous nature of the
sponge cells themselves have made automated counting


Page 1

MARINE INVERTEBRATE TISSUE CULTURE RESEARCH IN JAPAN

Akira Machii
Pearl Culture Laboratory
Yamakatsu Pearl Co., Ltd.
Kashikojima, Ago-cho, Shima-gun,
Mie-ken, 517-05
Japan

Although marine invertebrate tissue and cell culture
research is of great importance, there have been relatively
few researchers engaged in this field of study in most
countries. I consider it very fortunate for us to have this
opportunity to meet internationally and speak about these
important issues. I would like to introduce some back-
ground about marine invertebrate tissue and cell culture
research in Japan.

As you will see within the citations that accompany this
report, this area of study is still young. Machii’s work on
mantle tissue explant culture from the pearl oyster published
in 1974 opened research in marine Mollusca in Japan. Inthis
culture system, secretion of an organic substance (a kind of
abnormal pearl) caused the explant to change color from
milk white to dark brown. At the same time, amino acid
analysis from this secreted organic substance was also
performed by Yano and Machii (1975). In 1979, leyama et
al. reported mitoses in the cell cultures of gonad and mantle
tissue from the Pacific oyster, Crassostrea gigas. Develop-
ment and improvement of balanced salt solution and culture
media for both marine and freshwater. Mollusca were also
undertaken (Machii and Kawai 1980; Kawai et al. 1981;
Machii et al. 1989). Cell culture of a midgut gland from the
kuruma prawn (Crustacea) was enhanced (Machii et al.
1988). In this study, mitoses were observed, but subculture
was not obtained. In the past few years, due to the works of
Awaji and Machii (1989), Awaji et al. (1989), Itami et al.
(1989), Machii and Wada (1989), and Awaji and Durand
(1990), tissue and cell culture research on the pear! oyster
and the prawn has made further progress.

Through the Ministry of International Trade and Indus-
try, new and exciting research has been undertaken in
collaboration with major industrial firms of Japan’s Marine
Biotechnology Institute (Kamaishi). The institute will at-
tempt to develop cell cultures from 39 species of marine
invertebrates (Coelentrata, Mollusca, Echinodermata,
Arthropoda, etc.) and from other marine plants and animals.
Other excellent work is now in progress by Awaji (1991) of
the National Research Institute of Aquaculture. His studies
concentrate on the improvement of cell dissociation and
purification of dissociated cells from outer epithelium of
mantle tissue in the pearl oyster.

REFERENCES CITED

Awaji, M. 1991. Separation of outer epithelial cells from
pearl oyster mantle by dispase digestion. Paper presented
at: 1991 World Congress on Cell and Tissue Culture, 16-
20 June 1991, Anaheim, CA. Available from: Yamakatsu
Pearl Co., Ltd., Mie-ken, 517-05, Japan.

Awaji, M.; Durand, P. 1990. Purification of outer epithelial
cells from pearl oyster mantle: material for in vitro studies
on neurohormonal control of shell formation. Paper
presented at: Symposium on Molluscan Neurobiology,
Amsterdam, The Netherlands. Available from: Yamakatsu
Pearl Co., Ltd., Mie-ken, 517-05, Japan.

Awaji, M.; Machii, A. 1989. A method for separation of
outer epithelial cells from pearl oyster (Pinctada fucata
martensii) mantle. Paper presented at: 62d Meeting of the
Japanese Tissue Culture Association, Yokohama, Japan.
Available from: Yamakatsu Pearl Co., Ltd., Mie-ken,
517-05, Japan.

Awaji, M.; Machii, A.; Nakamura, H.K.; Wada, K.T. 1989.
Characterization of a zoosporic fungus appearing in pri-
mary cultures of the kuruma prawn midgut gland. Bull.
Nat. Res. Inst. Aquacult. 15: 19-27.

-leyama, H.; Takubo, M.; Moribe, S. 1979. Explant culture

of the oyster, Crassostrea gigas. Mem. Ehime Univ. Nat.
Sci. Ser. B (Biol.) 3: 1-4.

Itami, T.; Aoki, Y.; Yu, Y.; Takahashi, Y. 1989. In vitro
maintenance of lymphoid organ in kuruma shrimp Penaeus
Japonicus. Nippon Suisan Gakkaishi 55(12): 2205.

Kawai, S.; Machii, A.; Kitamura, S. 1981. Tissue culture of
aquatic Mollusca. Annu. Rep. Osaka City Inst. Publ.
Health Environ. Sci. 44: 64-72.

Machii, A. 1974. Organ culture of mantle tissue of the pearl
oyster Pinctada fucata (Gould). Bull. Nat. Pearl Res. Lab.
18: 2111-2117.

Machii, A.; Kawai, S. 1980. Physiological saline for aquatic
Mollusca and maintenance of the tissue in vitro. Paper
presented at: 50th Meeting of the Japanese Tissue Culture
Association, Tokyo, Japan. Available from: Yamakatsu
Pearl Co., Ltd., Mie-ken, 517-05, Japan.

Machii, A.; Wada, K.T. 1989. Some marine invertebrate
tissue culture. In: Mitsuhashi, J., ed. Invertebrate cell
system applications. Vol. II. Boca Raton, FL: CRC Press;
p. 225-233.


Page 4

Rosenfield, A. 1965. Maintenance of oyster tissue in vitro.
Am. Malacol. Union Inc. Annu, Rep. 1965: 30.

Stephens, E.B.; Hetrick, F.M. 1979. Decontamination of
American oyster tissues for cell and organ culture. Tissue
Cult. Assoc. Man. 5: 1029-1031.

Tripp, M_R.; Bisignani, L.A., Kenny, M.T. 1966. Oyster
amoebocytes in vitro. J. Invertebr. Pathol. 8: 137-145,

Marine Crustaceans

Brody, M.D.; Chang, E.S. 1989. Development and utiliza-
tion of crustacean long-term primary cell cultures:
ecdysteroid effects in vitro. Invertebr. Reprod. Dev. 16:
1-3.

Chang, E.S.; Brody, M.D. 1989. Crustacean organ and cell
culture. In: Maramorosch, K.; Sato, G.H., eds. Advances
incell culture. Vol.7. Chicago, IL: Academic Press; p. 19-
86.

Chen, S.N.; Jong, K.J.; Kou, G.H. 1988. Cell cultures from
hematopoietic tissue and ovary of penaeid shrimp, Penaeus
monodon. In: Kuroda, Y.; Kurstak, E., Maramorosch, K.,
eds. Invertebrate and fish tissue culture. New York:
Springer-Verlag; p. 195-198.

Chen, S.N.; Jong, K.J.; Kou, GH. 1989. Cell cultures
derived from tissues of the penaeid shrimp, Penaeus
penicillatus, and hard clam, Meretrix lusoria. In:
Mitsuhashi, J.,ed. Invertebrate cell system applications.
Vol. II. Boca Raton, FL: CRC Press; p. 254-262.

Lightner, D.V. 1988. Diseases of penaeid shrimp. In:
Sindermann, C.J.; Lightner, D.V., eds. Disease diagnosis
and control in North American marine aquaculture. 2nd
ed. Amsterdam, The Netherlands: Elsevier Scientific
Publishing Co.; p. 6-128.

Luedeman, R.A.; Lightner, D.V. In press. Development of
an in vitro primary cell culture system from the penaeid
shrimp, Penaeus stylirostris and Penaeus vannamei.
Aquaculture.

Other Marine Invertebrates -- Tunicates

Raftos, D.A.; Stillman, D.L.; Cooper, E.L. 1990. In vitro
culture of tissue from the tunicate Styela clava. In Vitro
26: 962-970.

Neoplasms

Dawe, C.J.; Harshbarger, J.C., editors. 1969. Neoplasms
and related diseases of invertebrates and lower animals.
Nat. Cancer Inst. Monogr. 31; 772 p.

Dawe, C.J.; Harshbarger, J.C.; Kondo, S.; Sigamura, T.;
Takayma, S., editors. 1981. Phyletic approaches to
cancer: proceedings of the 1 1th International Symposium
of the Princess Takamatsu Cancer Research Fund, Tokyo,
1980. Tokyo, Japan: Japanese Scientific Societies Press;
400 p.

Elston, R.A.; Kent, M.L.; Drum, A.S. 1988. Progression,
lethality, and remission of hemic neoplasia in the bay
mussel, Mytilus edulis. Dis. Aquat. Org. 4: 135-142.

Farley, C.A. 1989. Selected aspects of neoplastic progres-
sion in molluscs. In: Kaiser, H.E., ed. Comparative
aspects of tumor development. Vol. 5. Cancer growth and
progression. Dordrecht, The Netherlands: Kluwer Aca-
demic Publishers; p. 24-31.

Farley, C.A.; Sparks, A.K. 1969. Proliferative diseases of
hemocytes, endothelial cells, and connective tissue cells
in molluscs. Bibl. Haematol. 36: 610-617.

Lightner, D.V.; Redman, R.M.; Bell, T.A.; Brock, J.A.
1984. Anidiopathic proliferative disease syndrome of the
midgut and ventral nerve in the kuruma prawn, Penaeus
japonicus Bate, cultured in Hawaii. J. Fish Dis. 7: 183-
191.

Reinisch, C.L.; Charles, A.M.,; Troutner, J. 1983. Unique
antigens on neoplastic cells of the softshell clam Mya
arenaria. Dev. Comp. Immunol. 7: 33-39,

Scarpelli, D.G.; Rosenfield, A., editors. 1976. Molluscan
pathology. Mar. Fish. Rev. 38: 1-50.

Yevich, P.P.; Barszcz, C.A. 1977. Neoplasia in soft-shell
clams (Mya arenaria) collected from oil-impacted sites,
Ann, N.Y. Acad. Sci. 298: 409-426.

Biotechnology

Attaway, D.H. 1989. Marine biotechnology. In: National
Sea Grant College Program Annual Report; 16 p. Avail-
able from: National Sea Grant College Program, Silver
Spring, MD 20910.

Bols, N.C. 1991. Biotechnology and aquaculture: the role
of cell cultures. J. Biotechnol. Adv. 9: 31-49.