A Cellophane-Strip Technique for Culturing Tissue in Multipurpose Culture Chambers

A new technique for the cultivation of living tissues in the multipurpose culture chamber is described. This procedure employs strips of cellophane as the agent for anchoring tissue explants to the coverslip walls of the chamber and disposes of the time-honored plasma-clot technique. The primary advance embodied in this procedure lies in the fact that cells emigrating from so-cultured explants manifest themselves in a highly differentiated manner comparable to the cells of origin, whereas the outgrowth from the same types of tissue in plasma clots results in a more undifferentiated type of growth. Comparisons of outgrowths from embryonic thyroid, bone, and muscle (chicken) are photographically documented, and attention is called to certain cytochemical methods which further corroborate the differentiated quality obtained with the cellophane-strip technique.     

INDUCTION OF THE FROG HATCHING GLAND CELL FROM EXPLANTED PRESUMPTIVE ECTODERMAL TISSUE BY LICL

When cells of the superficial layer explanted from the presumptive ectoderm of a Rana japonica early gastrula embryo at stage 10 were cultured in standard salt solution for 4–7 days, they differentiated into cement gland cells (CGCs), cilia cells (CCs) and common epidermal cells (CECs). When, however, these explants were treated with LiCl and transferred to Barth's solution, hatching gland cells (HGCs) and pigment cells were induced.
The optimum condition for inducing differentiation of HGC was treatment with 70 mM LiCl for 6–8 hr at 18°C. The best ability to react to the HGC-inducing stimuli resided in the superficial layer of the dorsal presumptive epidermis of the embryo at stage 10. Upon repeated stimulation, explants from stage 8 embryos underwent differentiation into nerve and pigment cells, whereas those from stage 11 embryos differentiated into CCs and CECs. Under optimum conditions, the total volume of HGCs induced amounted to about 70% of the explanted tissue. The culture media from LiCl-induced HGCs showed an apparent jelly-digesting activity, strongly indicating that the cells were functionally identical with those differentiated in situ .     

Melanogenesis of avian neural crest cells in vitro is influenced by external cues in the periorbital mesenchyme

Avian melanoblast differentiation was studied by explantation of the neural tube and periorbital mesenchyme. Outgrowths from the mesenchymal explants consisted of a mixed population of melanocytes, melanoblasts and fibroblasts, whilst typical neural crest populations migrated from the neural tube explants. Cells that differentiated within explants of mesenchyme, produced elongate black eumelanosomes of normal ultrastructure which were identical to those found in the ocular connective tissues. However, melanoblasts that differentiated within outgrowths of mesenchyme or neural tube produced round brown melanosomes of highly abnormal ultrastructure. Some of these melanosomes contained a few disorganised melanosomal filaments whilst others had granular melanin with complete absence of filaments. This abnormality of phenotype was invariant over a range of culture conditions that modified cell behaviour, the timing of differentiation and the abundance of the pigmented cells. These experiments suggest that local factors in the mesenchyme are essential for the induction of melanogenesis in the presumptive connective tissue melanocyte.     

In vivo tissue engineering chamber supports human induced pluripotent stem cell survival and rapid differentiation

Pluripotent stem cells are a potential source of autologous cells for cell and tissue regenerative therapies. They have the ability to renew indefinitely while retaining the capacity to differentiate into all cell types in the body. With developments in cell therapy and tissue engineering these cells may provide an option for treating tissue loss in organs which do not repair themselves. Limitations to clinical translation of pluripotent stem cells include poor cell survival and low cell engraftment in vivo and the risk of teratoma formation when the cells do survive through implantation. In this study, implantation of human induced-pluripotent stem (hiPS) cells, suspended in Matrigel, into an in vivo vascularized tissue engineering chamber in nude rats resulted in substantial engraftment of the cells into the highly vascularized rat tissues formed within the chamber. Differentiation of cells in the chamber environment was shown by teratoma formation, with all three germ lineages evident within 4 weeks. The rate of teratoma formation was higher with partially differentiated hiPS cells (as embryoid bodies) compared to undifferentiated hiPS cells (100% versus 60%). In conclusion, the in vivo vascularized tissue engineering chamber supports the survival through implantation of human iPS cells and their differentiated progeny, as well as a novel platform for rapid teratoma assay screening for pluripotency.     

Weak functional coupling of the melanocortin-1 receptor expressed in human adipocytes

The melanocortin (MC) receptor type-1 (MC1-R) is the only one of the five MC receptor subtypes expressed in human adipose tissue explants, human mesenchymal stem cells (MSCs), and MSC-derived adipocytes. Following our recent expression studies (Obesity 2007, 15, 40-49), we now investigated the functional role of MC1-R in these tissues and cells to deduce the coupling state of MC1-R to intracellular output signals in human fat cells and tissue. Expression of MC1-R by undifferentiated and differentiated MSCs was quantified by real-time TaqMan PCR. Intracellular output signals (cAMP, lipolysis, secretion of IL-6, IL-10, and TNF-alpha), as well as effects on the metabolic rate and proliferation of human MSCs were analyzed by standard assays, exposing undifferentiated and differentiated MSCs and, in part, human adipose tissue explants to the potent MC1-R agonist, [Nle(4), D-Phe(7)]-alpha-MSH (NDP-MSH). This agonist induced a weak cAMP signal in MSC-derived adipocytes. However, it did not affect lipolysis in these cells or in adipose tissue explants, nor did it modulate cytokine release and mRNA expression of IL-6, IL-8, and TNF-alpha upon LPS stimulation. In undifferentiated MSCs, NDP-MSH did not alter the metabolic rate, but it showed a significant antiproliferative effect. Therefore, it appears that MC1-R-effector coupling in (differentiated) human adipocytes is too weak to induce a regulatory effect on lipolysis or inflammation; by contrast, MC1-R stimulation in undifferentiated MSCs induces an inhibitory signal on cell proliferation.     

An In Vitro Test for Temperature Sensitivity and Resistance to Meloidogyne incognita in Tomato

An in vitro root explant tissue culture technique is described for determining susceptibility of tomato (Lycopersicon esculentum Mill.) breeding lines and cultivars to the root-knot nematode Meloidogyne incognita. Root explants were taken from 2-day-old seedlings cultured for 30 days at 28 C on Gamborg''s B-5 medium with or without nematode inoculum. The remaining portion of the root and stem from the excised root explants was transferred to soil in pots and grown to maturity in the greenhouse. In vitro root explants were evaluated for growth and occurrence of juveniles, adults, and egg masses. The regenerated plants were used to produce more seed, The proposed technique is simple, reliable, and adapted to routine screening of large numbers of F₁ and F₂ samples, and it utilizes less space than tests performed on intact plants in the greenhouse or growth chamber. Evidence is presented also on the breakdown of resistance to M. incognita under high temperature stress using this in vitro root explant technique.     

An improved organ culture method for adult mammalian lung

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.     

Effect of a short-term disturbance of the cell contacts on the mesodermal differentiation of clawed toad embryos

The differentiation of the presumptive mesoderm explants from different sectors of the early gastrula marginal zone was compared with that of the identical explants placed just after explantation into a medium free of divalent cations for 30 s. The development of the treated dorsal explants differed from that of control explants by the presence of well differentiated forebrain structures and the development of the explants from more ventral zones by a decrease in the occurrence of blood cells and nondifferentiated endoderm and an increase in occurrence of epithelioid structures, which form frequently deep invaginations. The shape of the treated explants was more organized and differentiated than that of the control explants. A conclusion has been reached that the development of epithelioid and forebrain structures in the explants is stimulated after a short-term disturbance of cell contacts.     

Weak Functional Coupling of the Melanocortin-1 Receptor Expressed in Human Adipocytes

The melanocortin (MC) receptor type-1 (MC1-R) is the only one of the five MC receptor subtypes expressed in human adipose tissue explants, human mesenchymal stem cells (MSCs), and MSC-derived adipocytes. Following our recent expression studies (Obesity 2007, 15, 40–49), we now investigated the functional role of MC1-R in these tissues and cells to deduce the coupling state of MC1-R to intracellular output signals in human fat cells and tissue. Expression of MC1-R by undifferentiated and differentiated MSCs was quantified by real-time TaqMan PCR. Intracellular output signals (cAMP, lipolysis, secretion of IL-6, IL-10, and TNF-α), as well as effects on the metabolic rate and proliferation of human MSCs were analyzed by standard assays, exposing undifferentiated and differentiated MSCs and, in part, human adipose tissue explants to the potent MC1-R agonist, [Nle⁴, D-Phe⁷]-α -MSH (NDP-MSH). This agonist induced a weak cAMP signal in MSC-derived adipocytes. However, it did not affect lipolysis in these cells or in adipose tissue explants, nor did it modulate cytokine release and mRNA expression of IL-6, IL-8, and TNF-α upon LPS stimulation. In undifferentiated MSCs, NDP-MSH did not alter the metabolic rate, but it showed a significant antiproliferative effect. Therefore, it appears that MC1-R–effector coupling in (differentiated) human adipocytes is too weak to induce a regulatory effect on lipolysis or inflammation; by contrast, MC1-R stimulation in undifferentiated MSCs induces an inhibitory signal on cell proliferation.     

Isolation and characterization of differentiated alveolar type II cells from fetal human lung

A method has been developed for isolating differentiated type II cells from human lung of 18-24-week gestation. The procedure involves an initial 4-day culture of lung explants in the presence of dexamethasone (10 nM) and triiodothyronine (2 nM). Type II cells (and fibroblasts) are isolated by trypsin digestion of the explants, two differential adherence steps and incubation overnight in primary culture. This method provides a high yield of type II cells ((50 +/- 15) X 10(6) cells/g wet weight of explant) with a purity of 85 +/- 5% in 16 experiments. The type II cells contain numerous perinuclear granules which stain darkly with toluidine blue and Papanicolaou stain; electron microscopy showed these inclusions to be lamellar bodies with tightly stacked, well defined lamellae. Type II cells, but not fibroblasts, were positive by immunofluorescence histology for surfactant apoprotein and binding of Maclura pomifera lectin which binds to the surface of type II but not type I cells in vivo. The rate of both [3H]acetate and [3H]choline incorporation into phosphatidylcholine (PC) was several-fold greater in type II cells than fibroblasts; the saturation of PC was 36.2 and 25.9%, respectively. Release of saturated PC was stimulated by terbutaline, the ionophore A23187, and tetradecanoyl phorbol acetate in type II cells but not fibroblasts. We conclude that differentiated type II cells can be isolated in relatively high yield and purity from hormone-treated explants of fetal human lung.     

Selection of a chemically defined medium for culturing fetal mouse small intestine

Summary We evaluated six commercially available tissue culture media in their capacity to support villi morphogenesis and enterocyte differentiation during duodenal development of the fetal mouse in vitro: McCoy's 5A, Medium 199, Swim's S77, Trowell T8, Leibovitz L-15, and RPMI-1640. The duodenal segments were resected at 15 d gestation, before the formation of intestinal villi. In the segments cultured with the first four media, no villi differentiated even at 72 h culture. The number of epithelial cells per transverse section of the explants did not increase at 24 h and thereafter the number of epithelial cells decreased, except with McCoy's 5A. With the Leibovitz and RPMI media, rudimentary villi differentiated at 24 h of culture and they attained their longest length at 48 h. With the RPMI medium, the number of epithelial cells doubled at 24 h of culture and with Leibovitz medium it doubled at 48 h. At the fine structural level absorptive cells remained poorly differentiated with all the media studied. Goblet cells were easily identified after 24 h culture; they had a well developed rough endoplasmic reticulum and numerous mucous granules. Endocrine cells differentiated in culture and they were loaded with secretion granules. It was concluded that the small intestine of the fetal mouse can be kept in organ culture for at least 72 h. Full maturation of absorptive cells seemed to require some additional factor(s) as they remained poorly differentiated with all the media studied. Because well differentiated endocrine cells were present in all the explants, it appeared that gastrointestinal hormones do not affect villi morphogenesis and absorptive cells differentiation. This investigation was supported by Grant MA-6069 from the Medical Research Council of Canada. Mr. P. A. Micheletti was supported by a studentship from the FCAC.     

Role of platelet factors and serum complement in growth of fibroblasts with high-affinity C1q complement receptors

Summary Cultures of human diploid fibroblasts are heterogeneous in that a subpopulation interacts via high-affinity receptors with the globular head regions of the C1q complement protein. Growth and synthetic properties of these cells are characteristic of cells residing in healing wounds and inflammatory lesions. At these sites, fibroblasts are exposed to regulatory molecules such as complement components and factors released from blood platelets. We assessed the effects of native complement proteins and platelet-derived factors on proportions and phenotypic stability of high-affinity and low-affinity receptor cells generated from explants of adult and embryonic connective tissue, using radioligand binding assays and immunofluorescence analysis by flow cytometry. Fibroblasts expressing high-affinity C1q receptors could be generated from explants only when factors from platelets were present in the medium; native complement proteins were not essential. High-affinity receptor cells could be generated only from tissue; they could not be generated by incubating cultures of the low-affinity receptor phenotype in medium containing platelet-derived factors. High-affinity receptor cells, once established from explants in the presence of platelet-derived factors, persisted through many replications in the absence of platelets. We obtained the same fibroblast phenotypes from embryonic skin as from adult gingiva, but the proportion of high-affinity receptor cells from skin was much greater. We conclude that factors derived from platelets are essential for generating cultures containing fibroblasts expressing high-affinity C1q receptors, but not for their maintenance. High-affinity receptor cells may comprise a rapidly dividing subpopulation giving rise only to like progeny or to other, more differentiated cells. Supported by grants DE03301 and DE02600 from the National Institutes of Health, Bethesda, MD.     

Organ Culture of Foetal Rat Pancreas

The differentiation and growth of the foetal rat pancreas (20 days postcoitum) was studied in parabiotic organ culture with foetal adrenal tissue. In such co-cultures, characteristic pancreatic morphology was preserved and further acinar cell differentiation was fostered. Acinar cells continued to represent about 65% of the total explant volume following short-term incubation. The selective islet cell proliferation, previously observed in control pancreatic explants cultured alone, did not occur when adrenals were co-cultured. In addition, the amylase content of the incubation media and of the explanted pancreatic tissue remained high with adrenal co-culture, while the insulin content of the media and of the explanted tissue was markedly suppressed when compared to control pancreatic explants cultured alone. The effects of the adrenal in maintaining the differentiated acinar component of the pancreas and suppressing media insulin concentration diminished over extended incubation. The addition of adrenals to culture of foetal pancreatic explants after 6 days of control culture (at a time when differentiated acinar cells were not identifiable in the explant) did not result in redifferentiation of the acinar component, but did markedly depress media insulin content. Removal of adrenals after 4 days of co-culture resulted in an immediate rise in media insulin concentration and a rapid decline in pancreatic acinar mass. An adrenal-exocrine pancreatic axis is proposed and it is suggested that foetal adrenal secretions may play an important role in the development of the exocrine pancreas in vivo as well as in vitro.     

人脐带间充质干细胞的分离培养与鉴定简

目的：建立并优化人脐带间充质干细胞分离纯化方法,并对其表面标志与多向分化潜能进行鉴定。方法：收集健康足月产胎儿脐带组织,采用组织块贴壁法进行原代培养,流式细胞仪对其表面标志进行检测,通过向成骨成脂分化对其多向分化潜能进行鉴定,RT-PCR对其干细胞特性基因Oct4、Nanog、Sox2、Nestin进行检测。结果：采用组织块贴壁法可在2周左右获得大量间充质干细胞,培养的细胞经流式细胞仪检测,高表达CD29、CD44、CD105、CD106,低表达CD34、CD45;经成骨成脂诱导2周后可分化为成骨细胞和成脂细胞,RT-PCR检测发现原代细胞表达Oct4、Nanog、Sox2、Nestin基因。结论：人脐带间充质干细胞可在体外扩增培养,具有多向分化潜能,可作为组织工程种子细胞来源。     

The Culture of Excised Tissue 3Lilium speciosumThun

Explants from the bulb scales of Lilium speciosum were culturedin vitro, where they proliferated and differentiated to regeneratebulblets in 15–16 weeks. Morphologically, regenerationtakes place from mesophyll tissue, buds arising from more superficiallayers, and roots from deeper layers. The regenerative capacity of explants has been shown to be seasonaland of a localized nature, basal explants regenerating freely,apical explants never regenerating. The seasonal response maybe correlated with the vegetative state of the plant at thetime of excision.     

Embryonic induction and cation concentrations in amphibian embryos

Explanted ectoderm from early gastrulae of Triturus alpestris was treated with the Na-K ionophore gramicidin (10(-9) to 10(-5) M) and the Ca-ionophore A 23187 (10(-7) to 10(-5) M). The ectoderm developed almost exclusively to atypical epidermis as in the control explants. When the ectoderm was treated with ouabain (10(-4) M), intracellular Na+ increased about 4.4-fold and K+ was reduced by half. Mesenchyme cells in small number differentiated in about 40% of the ouabain-treated explants. The time course of total Na+ and K+ ion concentrations was measured over a period of 72 h in ectoderm of T. alpestris after induction with vegetalizing factor and in control explants. In the first 15 h after explantation, no significant differences between control and induced explants were found. Thereafter, the steady state concentration of K+ decreased in the induced explants, whereas the steady-state concentration of Na+ slightly increased. The membrane resting potential recorded intracellularly of ectoderm sandwiches from early gastrula stages was found to be -41.3 mV in control and -59.3 mV in induced explants. From the specific conductances and permeabilities of non-induced and induced cells it is concluded that the induction process leads to a differentiation of the cell membrane, which acquires the characteristics of ionic selectivity. Ectoderm from Ambystoma mexicanum forms neural or neuroid tissue, mesenchyme and melanophores after explantation in salt solution in up to 50% of the explants without any additions. Isolated Ambystoma ectoderm is therefore not suitable for test experiments.     

Comparison of induction during development between Xenopus tropicalis and Xenopus laevis

Several in vitro systems exist for the induction of animal caps using growth factors such as activin. In this paper, we compared the competence of activin-treated animal cap cells dissected from the late blastulae of Xenopus tropicalis and Xenopus laevis. The resultant tissue explants from both species differentiated into mesodermal and endodermal tissues in a dose-dependent manner. In addition, RT-PCR analysis revealed that organizer and mesoderm markers were expressed in a similar temporal and dose-dependent manner in tissues from both organisms. These results indicate that animal cap cells from Xenopus tropicalis have the same competence in response to activin as those from Xenopus laevis.     

Development of an in vitro culture method for cells and tissues from the zebra mussel (<Emphasis Type="Italic">Dreissena polymorpha</Emphasis>)

Despite the successful transfer of mammalian in vitro techniques for use with fish and other vertebrates, little progress has been made in the area of invertebrate tissue culture. This paper describes the development of an in vitro technique for the culture of both cells in suspension and tissue explants from the gill, digestive gland and mantle of the zebra mussel (Dreissena polymorpha) and their successful maintenance in culture for up to 14 days. Cell suspensions from the gills and digestive gland were the most successful technique developed with viability >80% maintained for up to 8 days in culture, suitable for use in short term toxicity tests. Tissue explants from the mantle were also maintained in culture for up to 14 days. This paper describes the challenges involved in the development of a novel in vitro culture technique for aquatic invertebrates.     

An autoradiographic study of the role of satellite cells and myonuclei during myogenesis in vitro

Satellite cells and myonuclei of neonatal rat muscles were differentially labeled with 3H-thymidine according to the procedure of Moss and Leblond (1971). Minced muscles fragments containing either labeled satellite cells or labeled myonuclei were cultured until multinucleated myotubes grew out from the explants. Reutilzation of isotope released from degenerating nuclei was competitively inhibited by using a culture medium containing excess (0.32-0.41 mM) cold thymidine. after an 8-10 day growth period, the explants were fixed and prepared for autoradiographic (ARG) examination to determine whether labeled satellite cells or myonuclei had contributed to the myonuclear population of the developing myotubes. Counts were made of the number of labeled myotubes in the explants and compared with the number of labeled satellite cells and myonuclei in samples of the original muscle tissues fixed at the time of explantation. The original muscles showed a mean satellite cell labeling index of 51.7% and gave rise to myotubes with a mean labeling incidence of 40%. In contrast, myonuclear labeling in the original muscle tissues showed no correlation with subsequent myotube labeling. Only 3.4% myotube labeling was found in explants in which over 30% of the original tissue myonuclei had been labeled. Under conditions controlled for isotope reutilization, these observations confirm results of in vivo ARG studies indicating that satellite cells are the only significant source of regenerating myoblasts in injured muscle tissue.