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Protocol no. 29
HUMAN THYROID CULTURE
This method enables the culturing of thyroid
cells without loss of differentiation and medium change. It is potentially
useful for the long-term study of drug effects on the thyroid gland.
CONTACT
Dr. C. Mothersill Radiobiology Research Group
Nuclear Energy Board Clonskeagh Road Dublin 14 Ireland
RATIONALE
Human thyroid cells obtained during surgery can
be maintained in culture for periods of up to two months without losing
morphological or functional differentiation. In the clinical situation
the thyroid may be exposed to long-term drug or radiation treatment which
may have adverse effects on the functioning of the thyroid gland. These
adverse effects can be assessed in culture by studying morphological and
biochemical changes after exposure to test chemicals, cytotoxins or radiation,
and extrapolated to the likely toxicity in man.
BASIC PROCEDURE
Sections of human thyroid are incubated in a
trypsin/collagenase solution. The resulting supernatant is filtered and
centrifuged twice. The cells are collected and resuspended in growth medium
and any undigested thyroid tissue is re-incubated in the trypsin/collagenase
solution on two further occasions. Each supernatant is filtered and centrifuged.
The digests are pooled and plated out in flasks containing Eagle's medium.
The cultures are incubated for 48 hours. The cultures are then exposed
to test chemicals and morphology, epithelia cell growth and biochemical
parameters are assessed. N.B. If the tissue sample is small, incubate with
the trypsin/collagenase solution and then plate directly into a flask.
After 48 hours, expose the cultures to test chemicals as above.
CRITICAL ASSESSMENT
A long-term culture system for sheep thyroid
has been established which retains many of the characteristic functional
and morphological features of the gland. The human thyroid culture has
been adapted from this sheep culture with only minor modifications. Some
morphological differentiation time discrepancies occur. Follicles develop
in sheep cultures in 5-8 days and in human cultures after 15-20 days, undifferentiated
areas are more common in human cultures and are visualised as patches of
epithelial cells devoid of follicles. The unusual glucose and lactate metabolism
of the sheep system permits a prolonged culture period. Glucose is rapidly
metabolised to lactate, and then the lactate is utilised by the cultures
over their remaining life-span. Exhaustion of lactate in the culture medium
coincides with cell death, but the latter can be delayed by adding concentrated
glucose to the medium just before this occurs. The metabolism of glucose
to lactate, and subsequent lactate utilisation, follows the same pattern
in human cultures, but at a much slower rate due to the lesser degree of
differentiation (lactate use correlates strongly with morphological differentiation).
The major factor in establishing a human thyroid culture is the amount
and character of healthy tissue obtained. The best cultures are from 5-10g
samples of thyroid tissue, although even samples of 0.05g have been cultured.
The slower rate of differentiation of the human culture system is advantageous
when long-term studies of drugs or radiation effects on the human thyroid
are required. The test chemical can be added directly above the differentiated
monolayer without disturbing the media or the degree of differentiation
that occurs. Although human thyroid cultures have been established and
utilised by other scientists, they have been short-term systems or subcultures
maintained by the use of hormones or growth enhancers. In general these
have been used to study the biochemical behaviour of the cultures in the
short-term or in the characteristics of the subculture. In these cases
the primary culture was not maintained for more than 7 days. This culture
system correlates well with the in vivo situation. Thyroid cultures have
a limited life span in humans (15-20 doublings) which equates to the deterioration
of cells in culture after the third or fourth subculture. The best endpoints
for determining the cells are functioning correctly are the T4 assay or
125I trapping ability. The thyroid culture shows a progressive loss of
differentiation after repeated subculture. It is postulated that this may
be due to the effects of the trypsination, which causes the release of
a receptor component into the medium which binds thyrotropin. This receptor
is regenerated when the cells are replated but it is thought that the regeneration
declines after repeated subcultures. Fibroblast contamination can occur
in cultures derived form low initial cell numbers. Seeding high numbers
of cells probably inhibits the proliferation of any fibroblasts present.
A large number of scientists use whole animals or animal cell culture systems
(mostly rodent) which have limited use in relation to the study of human
disease and toxicity. The heterogeneity of the source material, in terms
of genetic make-up and previous history of cytotoxic insult, is a disadvantage
in relation to the development of a standardised routine test system.
TEST STATUS
In-house development
REFERENCES
- Gutmann, I. & Wahlefel, A.W. (1974) Enzymatic
measurement of lactic acid in samples of serum and plasma. In: Methods
of Enzymatic Analysis (ed. Bergmeyer, H.U.) Academic Press, New York, pp
1464-1467
- Mothersill, C., Seymour, C., Moriarty, M.J. &
Cullen, M.J. (1985) Long-term culture of differentiated human thyroid tissue.
Acta Endocrinologica, 108, 192-99
- Schmidt, F.H. (1961) Enzymatic determination
of glucose in biological samples. Klin. Wochenschr. 39, 1244
IP-29 © November 1990
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