J. Embryol. exp. Morph. Vol. 20, 1, pp. 73-80, August 1968
With 2 plates
Printed in Great Britain
73
Growth and differentiation of Tubularia cells in
a chemically defined physiological medium
By ALLISON L. BURNETT1 , FAITH E. RUFFING 1 ,
JUNE ZONGKER1 & ANNA NECCO2
From the Stazione Zoologica, Napoli, Italia
Although hydroids have proven valuable experimental animals for studies
involving polarity and regeneration, they have not been extensively used by
chemical embryologists studying control mechanisms in differentiation. Ideally,
hydroids should be valuable tools for such a study. Their morpohology is
relatively simple since they are diploblastic; their cells achieve a high degree of
specialization (cnidoblasts, nerve cells, gland and mucous cells); cell differentiation (and morphogenesis) from a reserve stock of interstitial or i-cells is
rapid; and many species can be cultured in large numbers under controlled
environmental conditions.
Probably one of the reasons for this lack of attention is that no one has
succeeded in cloning cells of a particular type in a chemically defined medium.
In vivo systems, mainly because of their impermeability to most exogenous
materials with molecular weights over 200, have not proven to be especially
reliable.
Our objective in the present study was to devise an in vitro system which
satisfied the following conditions:
(1) A chemically defined physiological solution: by chemically defined we
mean a solution which does not contain sera or proteins and one with a precise
ionic environement. (2) Retention of morphology by cultured cells: in order to
understand factors controlling differentiation, the origin of the cells in culture
must be known. Extensive morphological dedifferentiation in culture makes
this identification impossible. (3) I-cells pursue normal differentiation pathways:
this population of cells may prove to be most valuable in studies on differentiation because normally they are pluripotent; that is, the same cell can differentiate in various directions depending upon environmental factors (Burnett, 1966).
(4) Occurence of cell division in all types of cells which divide in the in vivo
1
Authors'1 address: Department of Biology, Developmental Biology Center, Western
Reserve University, Cleveland, Ohio 44106, U.S.A.
2
Author's address: Stazione Zoologica, Napoli, Italia.
74
ALLISON L. BURNETT AND OTHERS
system. (5) The solution should support the existence of single cells making
cloning a possibility.
The present paper will describe the behavior of explants from the stem of
Tubularia cultured in a chemically defined medium. The behavior of the cells
will be examined in light of the foregoing requisites and the advantages of the
present system will be discussed in terms of previous work dealing with in
vitro hydroid systems.
Although we have examined well over 100 cultures at various time intervals
both as living and stained preparations, the following observations must remain
preliminary. To date, only six cultures have been analysed quantitatively. In
these cultures cell density, number of divisions of each cell type, and total
numbers of each cell type were recorded. Two cultures were chosen from the
24, 48, and 72 h stages and 2000 cells in each culture were recorded at random.
Although these counts are not significant, they will be referred to from time to
time to provide a rough index of cellular behavior.
Since our animals were collected daily from the Bay of Naples, they were
not in the same stages of development and there was no way of knowing if they
were in similar physiological states. Although we tried to select healthy animals
that appeared to be in the same developmental state, we could in no way be
assured that we were working with a homogeneous population. Nevertheless
90 % of the cultures behaved in a remarkable uniform manner. The remaining
cultures showed interesting but deviant behavior and will not be discussed until
more is known concerning factors responsible for this behavior.
MATERIALS AND METHODS
Our experimental animal was Tubularia sp. (At the present time there is a
controversy concerning the species of Tubularia found in the Bay of Naples.
Both larynx and crocea have been claimed to be the Naples species.) Animals
were collected daily from the Bay of Naples and were fed Artemia nauplii on
arrival in the laboratory. The animals were then cleaned of debris, washed, and
placed in Instant Ocean (synthetic sea salts used to prepare this artificial sea
water can be obtained from Aquarium Systems, Inc., 1450 E. 289th St., Wickliffe, Ohio) containing 0-03 % kanamycin. Animals to be employed for in vitro
experiments were starved for 48 h before use.
The preparation of hanging drop cultures was as follows. Animals were
placed in a Petri dish containing Instant Ocean adjusted to pH 3 with HC1. The
hydranth was excised at its junction with the stem. The coenosarc was forced
from the stem by sliding microforceps along the perisarc. The proximal 15 mm
of the coenosarc was excised and placed in a depression slide. The Instant
Ocean was then drawn off and replaced with a physiological solution developed
by A. Necco. This solution, designated Necco's solution, is composed as shown
on opposite page.
Tubularia cells in vitro
g/i.
NaCl
KC1 1
CaCL
MgCl 2 .6H 2 O
KH 2 PO 4
Na,HPO 4
75
g/i.
25-800
0-644
0-644
3080
0020
0080
NaHCO 3
Na2SO4
Na-pyruvate
Na-fumarate
L-glutamine
Phenol red
0-200
0-920
1-500
1050
1090
0020
pH of solution, 7-5; antibiotics used in the solution: 0-300 g/1. kanamycin, 0100 g/1.
streptomycin, 0100 g/1. penicillin.
After 15 s the Necco solution is removed and replaced with fresh solution.
This washing is repeated six times. Cataract knives were employed to cut the
tissue lengthwise so that it would lie flat. The excised tissue was then transferred
to a coverslip and arranged so that the gastrodermis faced upwards. All excess
fluid was then withdrawn with a micropipette. One drop of Necco's solution
was then placed in a depression slide to prevent eventual evaporation of the
hanging drop. Care must be taken so that the drop in the depression doesn't
touch the hanging drop. After inversion of the coverslip over the depression
the chamber was sealed with a mixture of paraffin and Vaseline in equal proportions. The cultures were maintained in the dark at 18 °C.
Although it is not necessary to change the culture medium for the first 6 days,
the medium may be changed as follows. The coverslip is removed with a razor
blade and inverted. A drop of fresh medium is added to the tissue and then
drawn off. This process is repeated three times. Care must be taken not to
dislodge the attached tissue during the washing. All excess liquid is then removed from the tissue and the coverslip is placed over a clean depression slide
containing a fresh drop of culture medium.
All of the above procedure was carried out under sterile conditions. All
glassware was wrapped in heavy paper and placed in a 170 °C oven for 3 h
before use. Rubber stoppers and filters were autoclaved at 120 °C for 30 min.
Necco's solution was filtered through 3 G5 sintered glass filters. Surgical instruments were kept in 70 % alcohol and flamed before use.
Cultures were prepared for histological examination as whole mounts. Tissues
were fixed in Bouin, and stained in toluidine blue adjusted to pH8. They were
then dehydrated, cleared in toluene and mounted in Permount.
RESULTS
Cultures were observed over a period of 6 days. We have divided the developmental sequence into three major stages: (a) attachment, (b) migration and
division, (c) differentiation. Each of these stages will be described separately.
(a) Attachment
Attachment is accomplished by gastrodermal digestive cells within 4-6 h after
explanting (Plate 1,fig.1). Filopodial-like extensions often reaching for 50-100 fi
76
ALLISON L. BURNETT AND OTHERS
radiate from the periphery of the digestive cells. It is through these extensions
that the cells make firm attachment to glass. The peripheral digestive cells all
participate in the attachment process, although one side of the explant is sometimes favored over the other. A monolayer of digestive cells from one side of
the explant may extend eventually over a distance of several hundred microns,
while the other side, though attached, does not spread (Plate 1, figs. 2, 3).
Digestive cells are easily identified by their enclosed protein and lipid droplets,
carotenoid crystals, and large vacuoles (Plate 1, fig. 2). As the monolayer spreads,
it is difficult to determine if the spreading is due to active migration of the peripheral digestive cells, a general spreading of the cells throughout the explant,
or a combination of the two. It appears that the spreading is not due to cell
division because division in digestive cells is about one division per thousand
cells in the six cultures quantitated. On the other hand, we observed that in
explants where there was an extensive spreading of the monolayer there were
approximately 10 cells in a given area measured by a grid whereas in cultures
with limited spreading 100-150 digestive cells occupied the same area. Thus, it
appears that expansion or elongation of individual cells throughout the mass
contributes significantly to monolayer formation.
During the first 24 h in culture the spreading monolayer consists of only
digestive cells, the only cells which adhered quickly to glass. Only after the
monolayer is firmly established is there an invasion of epidermal cell types.
(b) Division and Migration
During the attachment period and spreading of the monolayer there are divisions in gastrodermal digestive cells, epithelial epithelio-muscular cells, and interstitial cells. These divisions continue through the 6-day period that we have
observed cells in culture (Plate 2, figs. 7-9). Digestive cells divide within the
monolayer and within the original explant, while epidermal cells divide only
within the explant for the first 24 h. We could not detect a significant difference
in division frequency between epidermal and gastrodermal cells, but more
counts will have to be made before this can be stated with certainty.
Division rates within the interstitial cells vary over wide ranges. Also, their
density within the explant is subject to much variation. For example, the number
of interstitial cells occupying equivalent areas in six cultures counted varied
from 100 to 500. Since interstitial cells in a particular nest often divide in synchrony it is not uncommon to see a nest of 4, 8 or even 16 or more cells undergoing simultaneous division (Plate 2, fig. 9). Dividing i-cells constitute from
1 % to 5 % of the total i-cell population.
After 24 h i-cells begin to invade the monolayer. At this stage the epidermal
cells also begin to extend over the monolayer. While the epidermal cells spread
as an intact sheet, i-cells actively migrate between the epidermal cells by means
of single blunt pseudopodia. Cnidoblast cells containing fully developed nematocysts also actively migrate between the epidermal cells as separate entities. Both
J. Embryo/, exp. Morph., Vol. 20, Part 1
PLATE 1
(All preparations are stained with toluidine blue.)
Fig. 1. Attachment of digestive cells of the gastrodermis 4 h after explantation. x 200
Fig. 2. Spreading of large, vacuolated digestive cells in monolayer during first 24 h. x 200
Fig. 3. Large monolayer occupied exclusively by digestive cells 24 h after explantation. x 200
Fig. 4. Invasion of interstitial cells into monolayer (arrows) 24-36 h after explantation. x 200
Fig. 5. Beginning of interstitial cell nests after epidermal epithelio-muscular cells and interstitial
cells have invaded the monolayer. Note that i-cells occur in nests of two cells (arrows), x 400
Fig. 6. Interstitial cell nests and nests of cnidoblasts (arrows) 3-6 days after explantation. x 400
A. L. BURNETT & OTHERS
facing p. 76
J. Embryo/, exp. Morph., Vol. 20, Part 1
PLATE 2
. im.
12
Fig. 7. Division of epidermal epithelio-muscular cell which has spread over the original
monolayer. Note the large vacuole which persists in the dividing cell and the two nondividing large interstitial cells at lower left, x 900
Fig. 8. Single large interstitial cell in division; note the high basophilia surrounding the
spindle. Note four large non-dividing interstitial cells (arrow), x 900
Fig. 9. Nest of ten interstitial cells (arrows). Eight cells in lower tier are in division while
upper two cells are non-dividing, x 400
Fig. 10. Nest of cnidoblasts (arrow) forming stenoteles in epidermis, x 400
Fig. 11. Tri-polar neurone over original monolayer. Arrows point to axons issuing from the
cell body. Note scattered undifferentiated interstitial cells, x 400
Fig. 12. Gland cell with swollen internal secretory droplets in monolayer of digestive cells, x 900
A. L. BURNETT & OTHERS
Tubularia cells in vitro
77
cell types possess a migratory rate of 5-10 /*/min. Although i-cells migrate as
independent entities, it is not uncommon to see several of them migrating as a
single column (Plate 1, fig. 6). Whether the lead cell weakens intracellular bonds
by forcing its way between cell membranes of epithelial cells and provides a
'pathway of less resistance' to cells which follow is not known. Another possibility is that a cell in migration may secrete materials externally, providing
biochemical landmarks which influence the migratory pathways of other i-cells.
Between 24 and 72 h, migrating i-cells begin to divide upon reaching the
monolayer. At 24-30 h most i-cells occur singly or in groups of two (Plate 1,
fig. 5). By 48 h nests of 4 and 8 cells are observed (Plate 1, fig. 6). Once an i-cell
divides it ceases migration and remains attached to its daughter-cell by rather
large intracellular bridges. These bridges are not apparent when the cells
occasionally separate after completing a division. As the cells move away from
one another long cytoplasmic links, 0-5 [i in diameter, are drawn out between
the cells. These links are so firm that one cell in migration may actually tow its
daughter-cell for a short distance.
(c) Differentiation
Once i-cells have divided to form nests of 8-32 cells there is a simultaneous
differentiation of the cells in the nest into a single type of cnidoblast (Plate 1,
fig. 6; Plate 2, fig. 10). Once nematocyst differentiation is complete, the cnidoblasts separate and begin to migrate over the original monolayer, where they
become concentrated near the periphery.
Some individual i-cells upon reaching the monolayer do not divide to form
nests but differentiate directly into neurones (Fig. 11). The neurones are either
bi-polar, tri-polar or multi-polar. Axons extending from a neurone may extend
for distances of 100 /i or further. The axons are often ruptured upon fixation,
making it difficult to trace them to their extremities.
In five cultures which, for some unexplainable reason, became highly contaminated with bacteria, i-cells differentiated into cnidoblasts and also into cells
identical to sperm. These 'sperm' bundles contained from 10 to over 100 cells.
They possessed a triangular head characteristic of the gametes of the species.
Interestingly enough, no nerve cells were observed in these preparations.
From 48 through 144 h many cultures showed an increase in the numbers of
gland cells in the gastrodermis (Plate 2, fig. 12). It was not possible to determine
with certainty the origin of these cells; that is, whether they arose from i-celis
which penetrated the gastrodermis or from divisions in pre-existing gland cells.
However, the fact that some gland cells were also observed in the epidermis,
clustered between i-cells which appeared to be in early stages of gland-cell
differentiation, suggests that they might have their origin in culture from epidermal cell types.
From day 3 to day 6 cell division and differentiation continue. If the culture
medium is not changed at this time, cells begin to slough from the periphery
78
ALLISON L. BURNETT AND OTHERS
and the culture disintegrates. Often during this period it is observed that one or
two i-cells within a nest do not differentiate into cnidoblasts (Plate 2, fig. 9).
Presumably these non-differentiating cells are the ones which serve as a reserve
for the formation of new nests.
It is of particular interest that some cell sloughing occurs from the periphery
of the culture through the 6-day culture period beginning at 24 h. The sloughed
cells include digestive cells, gland cells, i-cells, epithelial cells, and cnidoblasts.
These cells drift into the surrounding culture medium and remain healthy for
periods of up to 5 days (possibly longer because in this study all cultures were
fixed after 6 days). Individual cells when placed in fresh culture medium are
not viable, which suggests that after 24 h the medium has been conditioned in
some manner so that it will support the existence of individual cells. Future
experiments are planned in which conditioned medium will be regularly used in
an attempt to clone cells of various types.
DISCUSSION
Perhaps we can best sum up our results by stating that at this early point we
do not know any of the factors which control division, cell movement, attachment, or differentiation in the in vitro system. In most respects the cells behave
in culture in much the same way that they do in the normal animal. Divisions of
the cell types responsible for growth in intact animals (interstitial cells, digestive
cells, and epithelio-muscular cells) occur in culture. Cellular migrations (interstitial cells and cnidoblasts) occur in culture and can be easily observed in
hanging-drop preparations examined at a magnification of x 1000. None of the
differentiated cell types undergo a morphological change in culture and all stain
exactly as they do in whole animals. Finally, the normal differentiation sequence
of interstitial cells to cnidoblasts and neurones (and possibly sperm) occurs
in vitro.
For the foregoing reasons we believe the system is a reliable one, and, moreover, the effects of normal growth-promoting and growth-inhibiting materials
can be easily tested in this system (Burnett, 1966). Without a covering perisarc,
which is normally impermeable to exogenous materials, the cells can be treated
directly with drugs or natural growth promoters. Further, the initial attachment
and spreading of digestive cells to form a monolayer provides a simple method
of separating this single cell type from the rest of the explant. Since the monolayer is later invaded by interstitial cells which then differentiate into nerve,
the system might provide a useful tool to hydroid neurophysiologists, who are
never certain if they are recording directly from a nerve cell in the animal. By
inserting electrodes into the monolayer at various intervals it might be possible
to link the first occurrence of action potentials with the differentiation of interstitial cells into nerves.
Because of the thinness of the preparations it should be possible to prepare
Tubularia cells in vitro
79
time-lapse films of cell migration and differentiation. Also, the firm attachment
of the cells to glass prevents tissue movement by muscular contraction, thus
facilitating cinematography.
Perhaps one of the biggest advantages of this system over previous ones is
that the cells can be kept alive for extended periods in a chemically defined
medium. Sanyal & Mookerjee (1960), working with Hydra, studied cells in vitro,
but their culture medium was pond water and the cells only lived a few hours.
Philips (1961), using Edamine, an enzymic digest of lactalbumin, and artificial
sea water as a medium succeeded in keeping cells viable for long periods. However, in this system it was impossible to determine which cell type persisted. In
any case, the only cell types which survived seemed to be morphologically
identical. Li, Baker & Andrew (1963) reported a medium for maintaining Hydra
cells in vitro. Unfortunately, it was not possible to identify with certainty the
cell types which persisted in culture. Photographs accompanying the report
showing cells in division cannot be identified as to cell type. Finally, the medium
contains horse serum which renders it chemically 'undefined'.
The most successful culture system of hydroids to date was that of Martin &
Tardent (1963). They were able to keep Tubularia cells alive for extended periods
and the cells did not lose their morphological identity. They noted a differentiation sequence similar to the one which we have described for interstitial cells,
except that they did not observe differentiation into neurones or gametes. Although the authors put forth good evidence of an increase in cell numbers
during culture, they did not observe any mitotic figures. Perhaps mitoses were
not as abundant in their preparations as in ours and were overlooked. The major
drawback of their culture medium was its complex nature and that it employed
shrimp serum. Since this serum is not defined chemically it would eventually
prove to be an inadequate medium for our purposes.
The present medium is simple to prepare and provides a simpler system for
bioassay than those reported previously. Since acoelomates do not possess sera
per se one would naturally prefer the simplest medium supporting growth and
differentiation.
SUMMARY
A physiological solution which will support growth and differentiation in stem
explants of Tubularia is described. The medium differs from others described
for coelenterate culture in that it is known chemically and contains no serum or
proteins.
Tubularia stem tissue will spread in a monolayer within hours after explantation. The monolayer, consisting solely of digestive cells, is later overgrown by
the epidermis. Divisions in epidermal epithelio-muscular cells, interstitial cells,
and digestive cells are described. After invading the monolayer interstitial cells
differentiated into neurones or cnidoblast cells, thus preserving their normal
pathways of differentiation.
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ALLISON L. BURNETT AND OTHERS
RESUME
Croissance et differentiation de cellules de Tubularia
dans un milieu physiologique chimiquement defini
On decrit une solution physiologique permettant la croissance et la differentiation d'explants de pedoncule de Tubularia. Ce milieu differe de ceux deja
decrits pour la culture de Coelenteres en ce qu'il est chimiquement connu et ne
contient ni serum, ni proteines.
Le tissu de pedoncule de Tubularia s'etale en couche monocellulaire dans les
heures qui suivent Pexplantation. La couche monocellulaire, constitute seulement de cellules digestives, est recouverte plus tard par l'epiderme. On decrit
des divisions dans des cellules epidermiques epithelio-musculaires, des cellules
interstitielles et digestives. Apres invasion de la couche monocellulaire, les
cellules interstitielles se differencient en neurones ou cnidoblastes, suivant ainsi
leur voie normale de differentiation.
The present work was supported by a grant from the National Institutes of Health,
Bethesda, Maryland.
We wish to thank Dr Peter Dohrn, former director of the Stazione Zoologica, for his
generosity and encouragement during the course of this work. Also, we wish to thank the
staff of the Stazione for their excellent co-operation and efficiency during our year's stay.
REFERENCES
A. L. (1966). A model of growth and cellular differentiation in Hydra. Am. Nat.
100, 165-89.
Li, FU, BAKER, F. D. & ANDREW, W. (1963). A method for tissue culture of Hydra cells.
Proc. Soc. exp. Biol. Med. 113, 259-61.
MARTIN, R. & TARDENT, P. (1963). Kultur von Hydroiden-Zellen in vitro. Rev. suisse Zool.
70, 312-16.
PHILLIPS, J. H. (1961). Isolation and maintenance in tissue culture of coelenterate cell lines.
In The Biology of Hydra, pp. 245-51. University of Miami Press.
SANYAL, S. & MOOKERJEE, S. (1960). Experimental dissociation of cells from Hydra. Arch.
Entw. Mech. Org. 152, 131-5.
BURNETT,
{Manuscript received 5 October 1967, revised 4 December 1967)