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1.
An improved organ culture method for adult mammalian lung 总被引:1,自引:0,他引:1
Robert R. Guerrero Donald E. Rounds Jon Booher 《In vitro cellular & developmental biology. Plant》1977,13(8):517-524
Summary An improved method for maintaining adult rat lung in submerged organ culture is described in which the alveoli were inflated
with agar and 200-μm-thick hand-cut sections were mounted in Rose chambers. The conventional single-compartmented Rose culture
chamber was modified by adding a second chamber separated from the first by a gaspermeable membrane. One compartment functioned
as an air reservoir and the other housed the explants submerged in nutrient medium. Visking dialysis membrane used underneath
the explants prevented cell outgrowth and facilitated the exchange of nutrients and waste products at the glass-tissue interface.
Because of the excellent optical properties of the Rose chamber and the thinness of the explants, individual cell types can
be identified in the living tissue. The explants were studied with time-lapse cinematography, light microscopy, histology,
and with erythrosine B for dye exclusion. With this modified system the functional life span of the explants was increased
from 1 week to 1 month.
This study was supported by NHLBI Grant No. HL15098-05. 相似文献
2.
Summary A modified continuous-flow culture system (CFCS) was developed to maintain large explants of periodontium from adult mouse
in organ culture. The culture medium was stored in a reservoir outside of the incubator, pumped via polyvinyl tubing into
small glass culture chambers that were placed in the oxygenator and then collected in a waste flask. Medium was analyzed for
pO2, pCO2 and pH during the culture period. Three-molar and singlemolar explants of periodontium were maintained for 48 hr in the CFCS
at two different pO2 ranges: 100 to 120 mm Hg and 400 to 420 mm Hg. [3H]Proline was added 24 hr prior to sacrifice. Light-microscope morphological and radioautographic observations suggested that
cell viability and incorporation of [3H]proline, probably into newly synthesized protein, increased with an increase in pO2 and was related to a pO2 gradient extending from the periphery to the center of the explants. 相似文献
3.
Carl Monder Alena Hatle Coufalik 《In vitro cellular & developmental biology. Plant》1979,15(8):579-586
Summary Explants of fetal rat liver maintained in organ culture lost about 40% of their mass in 42 hr of incubation as a result of
decrease in blood cells and hepatocytes. Proteins from the cytosol and particulate elements of the tissue were found in the
culture medium. About 60% of this protein was degraded to peptides during culture. The transfer of malate and lactate dehydrogenases
from tissue to medium paralleled that of proteins. Glutamate dehydrogenase was lost from the mitochondria and in part leaked
through the cell membrane into the medium. Net loss of activity of the three enzymes occurred, probably as a consequence of
proteolytic degradation. Of 12 enzymes in liver tissue, the specific activities of eight—soluble malate dehydrogenase, glutamate
dehydrogenase, succinate dehydrogenase, phosphopyruvate carboxylase, hexosediphosphatase, glucose-6-phosphatase, tyrosine,
aminotransferase, and alanine aminotransferase—were unchanged or increased. Glycogen synthetase, aspartate aminotransferase,
pyruvate kinase, and lactate dehydrogenase decreased. Although changes in membrane permeability may have had some influence
on the results reported, the predominant effect was due to loss of protein from tissue as a result of discharge of total contents
of some of the cells into the medium. The residual explanted tissue retained its structural integrity. It is concluded that
fetal rat liver in organ culture provides a suitable model system for controlled studies with this organ in vitro.
This investigation was supported by grants from the National Institute of Child Health and Human Development (RO 1 HD09715),
National Cancer Institute (CA 14194), and United States Public Health Service General Research Support Grant RR 5589. 相似文献
4.
Long-term organ culture of mouse mammary gland 总被引:1,自引:0,他引:1
J. W. Harbell P. D. Bowman J. M. Shannon C. W. Daniel 《In vitro cellular & developmental biology. Plant》1977,13(8):490-496
Summary A method for maintaining mouse mammary gland in organ culture for periods of at least 30 days is described. Strips of the
number four mammary glands were cultured in individual tubes while fully submerged in Medium 199 supplemented with insulin,
aldosterone, ovine prolactin and bovine growth hormone. Exchange processes were aided by slowly rotating the tubes during
culture. Mammary tissue from midpregnant BALB/c and virgin GR/A mice was induced to undergo lobulo-alveolar development, secrete
and remain differentiated and metabolically active for the period of culture. Cells of both the ductal and alveolar epithelium
continued to synthesize DNA and divide. The submerged roller-tube culture allows the use of larger pieces of tissue than can
be accommodated in static culture, and the technique may prove applicable to the culture of a variety of tissues. 相似文献
5.
Violet Albert David Barkla Graeme P. Young 《In vitro cellular & developmental biology. Animal》1994,30(7):443-449
Summary To facilitate the study of regulators of differentiation and proliferation of small intestinal epithelium in the suckling
rat we have developed a serum-free organ culture system and used it to examine epithelial responsiveness to various regulatory
hormones. These hormones included the insulin-like growth factors (IGFs) whose action can be blocked by binding proteins in
serum. Jejunal explants from 5-day-old suckling rats maintained better brush border enzyme activity and better histology when
cultured under hyperbaric conditions for 24 h in serum-free Dulbecco’s modified Eagle’s medium/F12 medium than in RPMI 1640
plus 10% fetal bovine serum. Tissue responsiveness to various regulatory hormones was then tested in the serum-free medium.
Insulin had no significant effect on morphology, proliferation rate, or enzyme activity in 5-day explants after 24 h in culture.
However, insulin did increase lactase activity and induce the early appearance of sucrase in 10- and 12-day explants after
48 h culture. Dexamethasone increased specific activities of alkaline phosphatase (30%,P<0.001) and lactase (15%,P<0.001), and reduced shedding of alkaline phosphatase into the medium (P<0.001), in explants of 5-day-old rats cultured over 24 h. Dexamethasone combined with insulin had no obvious effect on the
rate of protein or DNA synthesis but did increase villus height (P=0.04) and crypt depth (P=0.001) and acted synergistically to further increase lactase activity above levels obtained by either alone. IGF-I and IGF-II,
des-(1–3)IGF-I, fibroblast growth factor (FGF), and growth hormone (GH) had no effect on morphology or biochemical activity
of explants after 24 or 48 h culture. In conclusion, histology, enzyme activity, protein, and DNA synthesis of suckling rat
jejunal explants were equivalent or better in serum-free than in serum-containing organ culture systems. Furthermore, biological
responsiveness was demonstrated by dexamethasone and insulin altering the explants morphologically or biochemically. None
of the IGFs or GH had any biological effects, raising doubts about their direct biological action on the developing intestinal
epithelium. 相似文献
6.
Whole retinae from Midas cichlids Cichlasoma citrinellum were maintained successfully in superfusion culture for 21 days post-removal and continued to exhibit normal light- and circadian-driven cone movements. 相似文献
7.
Metanephric development in serum-free organ culture 总被引:4,自引:0,他引:4
Ellis D. Avner Demetrius Ellis Thomas Temple Ronald Jaffe 《In vitro cellular & developmental biology. Plant》1982,18(8):675-682
Summary A new mouse metanephric organ culture system has been developed to study mammalian renal development. The system permits in
vitro organotypic differentiation in a serum-free, hormone supplemented medium consisting of Dulbecco’s minimal essential
medium (MEM) and Ham’s F12 medium supplemented with insulin, 5 μg/ml; PGE1, 25 ng/ml; T3, 3.2 pg/ml; hydrocortisone, 5 μg/ml; and transferrin, 5 μg/ml. In this system, metanephric development continues morphologically
beyond the S-shaped tubule stage. A well differentiated proximal tubule forms with a well defined brush border, specialized
intercellular connections, and an apical endocytic network. In addition, a unique devascularized glomerulus, with highly differentiated
podocytes surrounding areas of basement membrane, forms entirely from epithelial elements.
The present organ culture model goes beyond the limitations of previously described systems in that it does not require separation
of nephrogenic blastema from ureteric bud, nor require animal serum or nonspecific tissue extracts for metanephric development.
The model is thus suited for morphological, biochemical, and endocrinological study of normal and abnormal renal organogenesis. 相似文献
8.
James H. Resau Ph.D. Kosaku Sakamoto John R. Cottrell Carnell Newkirk 《Cell biology and toxicology》1986,2(3):401-415
Adult Syrian Golden hamster alimentary tract maintained as explants in organ culture was studied using the model system for hamster pancreas described by Resau et al. (1983a). Explants of esophagus, stomach, duodenum and colon were maintained in organ culture on Gelfoam® sponge rafts in a high-oxygen atmosphere with serum-supplemented CMRL-1066 medium. All of the tissues were observed to show evidence of sublethal acute cell injury during the first several days of culture. Subsequently, the epithelial tissues recovered from this injury, repopulated the denuded areas of the explants and replicated within the sponge matrix. Explants were maintained in a differentiated state for 30+ days and sampled for morphology to examine the process of cell injury, repair, differentiation and replication which occurs in mucosal epithelia. The percentage of basement membrane covered by epithelia in the explants from various tissues was compared to the level of LDH in the media to reveal the relationship between viability determined by biochemical and by morphological methods. Restitution of the mucosal surface occurred in all of the explants. We conclude that adequate populations of replicating cells are maintained within the epithelium of the hamster alimentary tract tissues in vitro so that restitution can occur through migration and subsequent differentiation of the epithelial cells within the mucosa of the explants.Abbreviations 4F-1G
4% formaldehyde 1% glutaraldehyde fixative
- LDH
lactate dehydrogenase
- OsO4
osmium tetroxide
- PAS/PLH
periodic acid, periodic acid Shiff lead hematoxylin stain 相似文献
9.
A. A. J. J. L. Rutten B. G. A. G. G. Béquet-Passelecq H. B. W. M. Koëter 《In vitro cellular & developmental biology. Plant》1990,26(4):353-360
Summary A new method was developed for rabbit skin organ culture. In a two-compartment model, skin discs were cultured on a Millicell-HA
insert unit with a microporous membrane which allows transport of culture medium via the dermis into the epidermis, whereas
the epidermal side remains free of direct contact with culture medium. In this relatively simple two-compartment organ culture
model, rabbit skin could be cultured for 7 d in RPMI 1640 medium supplemented with fetal bovine serum, or for 2 d in RPMI
1640 medium supplemented with cofactors. The histomorphology and ultrastructure of 7-d cultured rabbit skin discs was essentially
similar to that of freshly isolated rabbit skin. Keratinocytes in the stratum basale continued to divide during organ culture.
The terminal differentiation of the epidermis continued in vitro as was found by the presence of keratohyalin granules, the
intact stratum corneum, and keratin expression. Furthermore, glucose consumption continued until culture Day 7, but thereafter
it declined rapidly. Concomitantly, degenerative changes were found. At the end of the 7-d culture period the distance between
single dermal collagen fibrils had increased as compared to noncultured skin. This model of skin organ cultures can be used
to study biological processes, dermal toxicity, and penetration and metabolism of xenobiotics in intact skin. Furthermore,
within certain limits, processes responsible for repair and regeneration of damaged skin can also be studied in this model
because the rabbit skin can be cultured for 7 d.
The present study was financially supported by grants of Duphar B. V. (Weesp, Netherlands), the European Community, and the
Dutch animal welfare organizations Samenwerkingsverband van de Nederlandse Vereniging tot Bescherming van Dieren en de Nederlandse
Bond tot Bestrijding van de Vivisectie, Anti-Vivisectie Stichting en Stichting Schoonheid Zonder Wreedheid. 相似文献
10.
Ellis D. Avner William E. Sweeney Jr. Nicholas P. Piesco Demetrius Ellis 《In vitro cellular & developmental biology. Plant》1985,21(5):297-304
Summary In order to define humoral growth factors which may regulate mammalian renal development, the growth requirements of fetal
metanephric organogenesis were studied in serum-free murine organ culture. Metanephric growth, determined by cell proliferation
and protein content, and metanephric differentiation, determined morphometrically as epithelial glomerular formation, were
compared and contrasted following 144 hours of organ culture incubation in basal medium, basal medium supplemented with 10%
fetal bovine serum, and basal medium supplemented with various combinations of growth factors. The basal medium was composed
of equal volumes of Dulbecco's modified Eagle's medium and Hams' F-12 medium. Five humoral growth factors were studied in
the following concentrations: selenium, 6.8×10−9 M; insulin, 8.3×10−7 M; triiodothyronine, 2×10−9 M; transferrin, 6.2×10−8 M; and prostaglandin E1, 7.1×10−8 M. Results showed that transferrin and prostaglandin E1 were necessary for optimal growth in the system and that prostaglandin E1 was necessary for maximal metanephric differentiation. Such data provide guidelines for the creation of serum-free medium
for future fetal renal cell and tissue culture systems, and provide insight into the factors which may regulate normal and
abnormal renal embryogenesis and the reparative processes of renal hyperplasia and hypertrophy which follow renal injury.
A preliminary report of this work was presented at the Ninth International Congress of Nephrology, Los Angeles, June 1984.
These studies were supported in part by Basil O'Connor Starter Research Grant 5-349 from the March of Dimes Birth Defects
Foundation and New Investigator Research Award I-R23-AM34891-01 from the National Institute of Arthritis, Diabetes, and Digestive
and Kidney Diseases of the National Institutes of Health (Both to Dr. Avner).
Editor's Statement The determination of effects of extracellular components on the introduction and maintenance of differentiation
is an area for which serum-free culture techniques are particularly suited. The approaches described in this report utilize
morphometric techniques to quantitate differentiation in serum-free fetal organ culture in addition to standard methodologies
for assessing growth. The purely epithelial nature of the cultures used in these studies also provides some interesting advantages
in the design of experiments aimed, at determining the importance of cell-cell interactions at various stages in the differentiative
process. David W. Barnes 相似文献
11.
Paul A. Weinhold William E. Burkel Theodore V. Fischer Raymond H. Kahn 《In vitro cellular & developmental biology. Plant》1979,15(12):1023-1031
Summary The maintenance of adult rat lung explants in organ culture was assessed both morphologically and biochemically. A comparison
of several culture media indicated that Ham's F12K plus 0.1 μM dexamethasone, which maintained the explants for 14 days, was
superior. The ability of the explants to synthesize dipalmitoylphosphatidylcholine increased with the length of cultivation
to values greater than the noncultivated controls. The DNA content remained constants for 7 days, and a relatively normal
structural relationship between type I and type II pneumocytes was maintained. Explants cultivated in Ham's F12K without dexamethasone
did not maintain a histotypic morphology; the type II pneumocytes appeared to proliferate and the ability to synthesize phosphatidylcholine
decresed.
Support was given by NIH Grant HL19669 and from the Veterans Administration. 相似文献
12.
Organ explant culture of adult Syrian golden hamster pancreas 总被引:1,自引:0,他引:1
James H. Resau Eric A. Hudson Raymond T. Jones 《In vitro cellular & developmental biology. Plant》1983,19(4):315-325
Summary An organ explant culture system has been developed for long term maintenance of adult pancreatic tissue from the Syrian golden
hamster. Gastric and duodenal lobe explants of up to 0.5 cm2 size were placed in tissue culture dishes (60 mm2) on Gelfoam sponge rafts to which was added 5 ml of CMRL medium 1066 supplemented with heat inactivated newborn bovine serum,l-glutamine hydrocortisone, insulin, and antibiotics. Dishes were placed in a controlled atmosphere chamber, which was gassed
with 45% O2 50% N2, and 5% CO2 and incubated at 36.5°C. Viability of the tissues was determined by light and electron microscopy as well as by [3]thymidine incorporation. Explants were viable for up to 70 d. Zymogen granule-containing cells characteristic of acinar cells
and mucuscontaining cells characteristic of ductal cells were present throughout this period. However, endocrine cells were
only present for the 1st wk in culture.
This work was supported in part by National Cancer Institute Grant CA-19197-06 through the National Pancreatic Cancer Project
and is UMP contribution No. 950. 相似文献
13.
14.
Preston Lamberton Milton Lipsky Paul Mc Millan 《In vitro cellular & developmental biology. Plant》1988,24(6):500-504
Summary A new model for organ culture of endocrine tissue is described. Rat anterior pituitary fragments were cultured for 4 wk within
semipermeable polyurethane isocyanate hollow fibers. Growth hormone and prolactin, two of the anterior pituitary hormones,
were released into the medium during the entire culture period. Electron microscopy of the pituitary fragments after 2 wk
in in culture showed a rim of viable tissue in all specimens examined. Individual cells, from this outer rim, exhibited excellent
organelle preservation and numerous secretory granules. Experiments involving potassium depolarization and 10−6
M dopamine provided evidence for the normal responsiveness of the cultured pituitary tissue to both stimulatory and inhibitory
factors. These studies illustrate the potential utility of the described organ culture system for further investigations of
endocrine physiology. 相似文献
15.
Masayoshi Kumegawa Taishin Takuma Fumiko Murayama 《In vitro cellular & developmental biology. Plant》1976,12(10):718-728
Summary A new technique for organ culture which uses plastic culture chambers and the advantages of the cellophane-sheet technique
is described with the results of a study of cultivations of fetal mouse liver. Two chambers, each containing cells, were placed
in gas permeable roller tubes and rotated at 0.1 rpm in a CO2-air gassed incubator. The fetal mouse liver cells developed electron microscopic features similar to those of the in vivo
adult liver by 9 days of cultivation. The albumin content and tyrosine aminotransferase (TAT) activity were detected in the
cultivated liver. TAT activity was further induced by prednisolone. These results indicate the potential of this culture method
for the study of physiological and pathological processes.
This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture,
Japan and Science Technology Agency, Japan. 相似文献
16.
Summary The effect of variations in culture conditions on survival of fragments of mouse and rat descending colon in organ culture
was studied by morphological and functional criteria. A combination of conditions demonstrated to be beneficial permitted
maintenance for at least 35 days. These included: a gaseous environment of 95% O2:5% CO2, an attachment matrix consisting of a Metricel GA-4 membrane (pore size, 0.8 μ), intermittent exposure to the gas and fluid
phases by rocking in 5 ml medium and supplementation of the medium with 1.0 μM dexamethasone and 10% FBS. During this time,
the crypt structure of the mucosal epithelium was well preserved, and DNA synthesis in the crypts and mucin production in
the crypts and superficial epithelium continued. In addition, the synthetic hormone, pentagastrin, stimulated DNA synthesis
in the mucosal epithelium of mouse colon fragment in short-term organ culture.
This work was supported by Environmental Protection Agency Grant R803998-01-1 and National Cancer Institute Contract N01-CP-75952. 相似文献
17.
J. Feng A. H. Melcher D. M. Brunette H. K. Moe 《In vitro cellular & developmental biology. Plant》1977,13(2):91-99
Summary The method of Deutsch and Weeks was modified to provide a reliable and reasonably quick method for assaying the L-ascorbic
acid content of culture medium. The modified method was used to determine the decay of L-ascorbic acid under various conditions
of culture and the concentration of the vitamin in commercially prepared media. The half-life of L-ascorbic acid in a modified
New circulator gassed with 95% O2+5% CO2 was 1.5 hr.; and when gassed with 20% O2+5% CO2+75% N2, about 2 hr. In Petri dishes gassed with 20% O2+5% CO2+75% N2, the half-life of L-ascorbic acid was 0.9 hr. About 4% of the L-ascorbic acid was lost per day when medium was stored at
0°C and about 9% per day when stored at 5°C. When medium with an initial content of 300 μg per ml was stored at room temperature,
the half-life was found to be 15.5 hr. The L-ascorbic acid in five commercially available media, which contain the vitamin
in their formulations, was assayed immediately after their delivery to the laboratory. The values of L-ascorbic acid measured
in these media were in all cases far lower than prescribed. A continuous-flow organ culture system has been designed which
allows the provision of a relatively constant level of L-ascorbic acid to an explant by taking advantage of the slow oxidation
of L-ascorbic acid at 0°C. 相似文献
18.
David Cossar Jeanne Bell Malcolm Lang Robert Hume 《In vitro cellular & developmental biology. Animal》1993,29(4):319-324
Summary In utero, at around 23 wk gestation, the progenitor epithelium of distal airway differentiates into type I and type II pneumatocytes.
Human fetal lung organ cultures, as early as 12 wk gestation, have the competence to self-differentiate. Distal airway epithelial
immunoreactivity to cytokeratins CK 7,8, and 18 decreases with differentiation both in utero and in organ culture, whereas
reactivity to epithelial membrane antigen remains constant in both. As distal airways dilate, the mean percentage airspace
of fetal lungs in organ culture increases to 58%, equivalent to lung of gestation 26.0±7.3 wk. In organ culture, capillary
blood vessels, visualized by vimentin immunoreactivity, remodel and more closely approximate the epithelium but without direct
invasion. In utero, at 23 wk gestation, elastin appears as condensation around airways and forms a basis for secondary crests
which, by 29 wk gestation, evolve into alveolar septae. In organ culture, no elastin is deposited, no secondary or alveolar
crests form, and the lung retains a simple saccular structure. Differentiation of the terminal airway epithelium and mesodermal
maturational events to facilitate gas exchange, such as capillary invasion or secondary-alveolar crest formation, are almost
synchronous in human lung in utero but clearly dissociate in organ culture. 相似文献
19.
Mark W. J. Ferguson Lawrence S. Honig Pablo Bringas Harold C. Slavkin 《In vitro cellular & developmental biology. Plant》1983,19(5):385-393
Summary The development of the first branchial (mandibular) arch of the American alligator (Alligator mississippiensis) is studied in organ culture for the first time. There is extensive mandibular morphogenesis in vitro and the pattern of
the differentiated elements, bones, muscles, and cartilage, is comparable to that found during normal development in ovo.
In addition, we observe the development of paired lingual swellings and the formation of a small tongue consisting of fibrous,
lipid containing, and muscular tissues, as found in the normal tongue. Several culture variables are examined: (a) although
alligator embryos normally develop at 30°C, we successfully culture the mandibular rudiments with good short term (3 wk) results
at 37°CC; (b) at 30° C, we are able to culture arches for long term periods of 6 wk; (c) the mandibular arches develop well
in both serum containing medium and in a chemically defined medium free from added hormones. The reptilian mandibular arches
exhibit excellent, patterned, development and may have less stringent culture requirements than their avian and mammalian
counterparts.
Collection and specimen export was performed under U.S. Fish and Wildlife Permit PRT2-2511 and Northern Ireland Permit B/WL2/80
issued to M.W.J.F. Collaborative study was made possible by the 1981 award of a Research Travelling Scholarship to M.W.J.F.
from The Wellcome Trust, for which we are grateful. This work is supported by Grant 8113610 CB from the MRC of Great Britain,
Grant EP109/74/75 from the Northern Ireland Eastern Health & Social Services Board, and Grants DE-02848 and DE-03569 from
the National Institutes of Health, Bethesda, MD. 相似文献
20.
Lymphocyte activation gene-3 (LAG-3; CD223) is structurally similar to CD4 and binds to MHC class II with a 100-fold higher affinity than that of CD4. Soluble LAG-3 (sLAG-3Ig) might be useful for immunotherapy by inducing MHC class II-mediated cell activation. A new form of sLAG-3Ig was constructed containing a critical binding site (D1 and D2 region) to MHC class II, combined with a Fc portion of an immunoglobulin gamma1. After treatment of sLAG-3Ig in fetal thymic organ culture from DO11.10 transgenic mouse, CD4(+) T cell precursors were increased in the positive selection but not affected in the negative selection. Further analysis by treating sLAG-3Ig on thymic epithelial cells revealed that CD40 and MHC class II were up-regulated. These results may demonstrate that the treatment of sLAG-3Ig increases the precursor frequency of CD4(+) T cells by activation of thymic epithelial cells. 相似文献