Explant Culture Technique Research Articles

Culture of embryonic mouse cochlear explants and gene transfer by electroporation.

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development(1-8).

IL22 regulates human urothelial cell sensory and innate functions through modulation of the acetylcholine response, immunoregulatory cytokines and antimicrobial peptides: assessment of an in vitro model.

Human urinary disorders are generally studied in rodent models due to limitations of functional in vitro culture models of primary human urothelial cells (HUCs). Current HUC culture models are often derived from immortalized cancer cell lines, which likely have functional characteristics differ from healthy human urothelium. Here, we described a simple explant culture technique to generate HUCs and assessed their in vitro functions. Using transmission electron microscopy, we assessed morphology and heterogeneity of the generated HUCs and characterized their intercellular membrane structural proteins relative to ex vivo urothelium tissue. We demonstrated that our cultured HUCs are free of fibroblasts. They are also heterogeneous, containing cells characteristic of both immature basal cells and mature superficial urothelial cells. The cultured HUCs expressed muscarinic receptors (MR1 and MR2), carnitine acetyltransferase (CarAT), immunoregulatory cytokines IL7, IL15, and IL23, as well as the chemokine CCL20. HUCs also expressed epithelial cell-specific molecules essential for forming intercellular structures that maintain the functional capacity to form the physiological barrier of the human bladder urothelium. A subset of HUCs, identified by the high expression of CD44, expressed the Toll-like receptor 4 (TLR4) along with its co-receptor CD14. We demonstrated that HUCs express, at the mRNA level, both forms of the IL22 receptor, the membrane-associated (IL22RA1) and the secreted soluble (IL22RA2) forms; in turn, IL22 inhibited expression of MR1 and induced expression of CarAT and two antimicrobial peptides (S100A9 and lipocalin-2). While the cellular sources of IL22 have yet to be identified, the HUC cytokine and chemokine profiles support the concept that IL22-producing cells are present in the human bladder mucosa tissue and that IL22 plays a regulatory role in HUC functions. Thus, the described explant technique is clearly capable of generating functional HUCs suitable for the study of human urinary tract disorders, including interactions between urothelium and IL22-producing cells.

Serial explant culture provides novel insights into the potential location and phenotype of corneal endothelial progenitor cells

The routine cultivation of human corneal endothelial cells, with the view to treating patients with endothelial dysfunction, remains a challenging task. While progress in this field has been buoyed by the proposed existence of progenitor cells for the corneal endothelium at the corneal limbus, strategies for exploiting this concept remain unclear. In the course of evaluating methods for growing corneal endothelial cells, we have noted a case where remarkable growth was achieved using a serial explant culture technique. Over the course of 7 months, a single explant of corneal endothelium, acquired from cadaveric human tissue, was sequentially seeded into 7 culture plates and on each occasion produced a confluent cell monolayer. Sample cultures were confirmed as endothelial in origin by positive staining for glypican-4. On each occasion, small cells, closest to the tissue explant, developed into a highly compact layer with an almost homogenous structure. This layer was resistant to removal with trypsin and produced continuous cell outgrowth during multiple culture periods. The small cells gave rise to larger cells with phase-bright cell boundaries and prominent immunostaining for both nestin and telomerase. Nestin and telomerase were also strongly expressed in small cells immediately adjacent to the wound site, following transfer of the explant to another culture plate. These findings are consistent with the theory that progenitor cells for the corneal endothelium reside within the limbus and provide new insights into expected expression patterns for nestin and telomerase within the differentiation pathway.

Development of primary cell culture from the caudal fin region of snow trout Schizothorax plagiostomus (Heckel)

A primary cell culture has been established from the Snow trout Schizothorax plagiostomus. Primary cultures have developed from caudal fin tissues by explant culture technique. Cell outgrowth has been obtained from the caudal fin explant after 5-7 days of explant culture. The culture medium used for the experiment was Leibovitz-15 supplemented with 20% Fetal Bovine Serum. Radiation of cells from the explants started after 2 to 3 days. Both fibroblast like and epithelial like cells were noticed but fibroblast like cells dominated as the growth progressed.

Cultivation and Phenotypic Characterization of Rabbit Epithelial Cells Expanded Ex Vivo from Fresh and Cryopreserved Limbal and Oral Mucosal Explants

Purpose: To compare the morphology of cultured rabbit epithelial sheets and the expression of stem cells with differentiated cell markers of cultivated epithelial cells from fresh and cryopreserved limbal and oral mucosal biopsies.Materials and methods: Six New Zealand white rabbits were divided into two groups of three, from which limbal and oral mucosal biopsies were taken. Harvested tissues from each rabbit were brought to immediate cultivation, while another set of tissues was cryopreserved. Cultivation was performed by the explant culture technique using human amniotic membrane as a culture substrate, co-culturing with 3T3 fibroblasts and using the air-lifting method. Cells were cultured for three weeks; then cultured epithelial sheets were stained with hematoxylin–eosin and examined for expression patterns of p63, keratin 3 (K3) and connexin 43 (Cx43). Cryopreservation was carried out using the vitrification method. Tissues were preserved in liquid nitrogen using 25% dimethyl sulfoxide combined with 25% propylene glycol in Dulbecco’s Modified Eagle’s Medium containing 20% fetal bovine serum. After two months, the tissues were warmed, cultured and stained using the same processes as for fresh tissue cultures.Results: Cultivation of fresh limbal and fresh oral mucosal tissues showed epithelial stratification, with two to five cell layers. Immunohistochemical staining showed p63-positive cells in basal and intermediate cell layers. K3 staining was observed in cells in the suprabasal layer, while expression of Cx43 was scattered throughout all layers of the epithelia. All culture sheets expressed p63, K3 and Cx43 with the exception of one sheet from the oral mucosal culture that was p63-negative. Cultured epithelial sheets from cryopreserved tissues showed results similar to those from fresh tissue culture.Conclusions: This study found that cells in cultivated fresh limbal and oral mucosal tissues had similar morphology to cells in cultivated cryopreserved limbal and oral mucosal tissues, both containing a heterogeneous population of cells including stem cells and differentiated cells.

Attempt at conserving the genetic resources of Hu sheep by fibroblast line cryopreservation

Hu sheep is one of the most distinctive local sheep breeds in China, while investigations concerning its cell biology and molecular genetics are still scarce. To preserve the valuable genetic resources of the Hu sheep, we established a Hu sheep fibroblast line from 35 sheeps' ear marginal tissues using explant culture and cryopreservation techniques, and its biological characteristics were identified, including cell morphology, growth curve, karyotype, isoenzyme polymorphism, and expression of exogenous fluorescent genes. The results showed that the viability of the fibroblasts before freezing was 96.54 ± 3.22% and 92.36 ± 2.77% after thawing, and there was no microbial contamination and cross-contamination; the growth curve of Hu sheep ear marginal fibroblasts displayed an obvious “S” shape; all somatic chromosomes were acrocentric autosomes, and only the two sex chromosomes (XY) were submetacentric; the normal number of chromosomes (2n = 54) was observed in 96.56 ± 2.89% of the cells; and the transfer efficiencies of four fluorescent proteins could achieve 37.4% with an optimized ratio of plasmid and lipofectamine. It could be concluded that this study has not only provided the biological characteristics of Hu sheep at cellular level, but also makes a valuable contribution to the preservation of the genetic resources of the Hu sheep.

Unilateral Partial Limbal Stem Cell Deficiency: Contralateral Versus Ipsilateral Autologous Cultivated Limbal Epithelial Transplantation

To report the outcomes of autologous cultivated limbal epithelial transplantation using the healthy part of the affected eye or the fellow eye as a source of limbal stem cells in patients with unilateral, partial limbal stem cell deficiency (LSCD). Retrospective, nonrandomized, interventional case series. setting: L. V. Prasad Eye Institute, Hyderabad, India. study population: Patients with unilateral, partial LSCD who underwent autologous cultivated limbal epithelial transplantation between 2001 and 2011. intervention: The limbal biopsy was taken either from the healthy part of the limbus of the same eye (ipsilateral group) or from the healthy fellow eye (contralateral group). Cells were cultivated using a xeno-free explant culture technique, and cultivated cells were transplanted onto the affected surface. primary outcome measure: Success of cultivated limbal epithelial transplantation, defined as a completely epithelialized, avascular, and clinically stable corneal surface. Seventy eyes of 70 patients were studied. The mean follow up was 17.5 ± 7 months. In 34 eyes the limbal biopsy was taken from the ipsilateral eye and in the remaining 36 eyes from the contralateral eye. Clinical success was achieved in 70.59% of eyes in the ipsilateral group and 75% of eyes in the contralateral group (P = .79). Limbal transplant survival rates at the final follow-up visit were 65.1% ± 0.09% in the ipsilateral group and 53.6% ± 0.12% in the contralateral group (P = .74). Ocular surface restoration in partial LSCD is possible with cell-based therapy. Outcomes are similar irrespective of whether the limbal biopsy is taken from the healthy part of the ipsilateral eye or the contralateral eye.

The effect of basic fibroblast growth factor and adipose tissue-derived mesenchymal stem cells on wound healing, epithelization and angiogenesis in a tracheal resection and end-to-end anastomosis rat model

Background: This experimental study aims to investigate whether basic fibroblast growth factor, adipose tissue-derived from mesenchymal stem cells, or a combination of both, has an effect on wound healing, epithelization and angiogenesis in a tracheal resection and end-to-end anastomosis rat model. Methods: During the first phase of the study, mesenchymal stem cells were isolated by the primary explant culture technique from the abdominal adipose tissue of rats. When the cells became confluent, they were passaged and characterized by using immunofluorescence staining technique. The cells were cryopreserved for an in vivo application. The rats were divided into four groups, including: the basic fibroblast growth factor (group 1), the mesenchymal stem cells (group 2), the mesenchymal stem cells and basic fibroblast growth factor (group 3) and control group (group 4). The rats were sacrified at day 60 and the anastomosis was evaluated macroscopically for granulation tissue formation, the stenosis and presence of tracheocutaneous fistula formation, and also microscopically for stenosis, epithelium regeneration, inflammation, collagen formation and neovascularization. Results: The inflammation was significantly lower in the study groups (p=0.004, p=0.014, p=0.004), whereas the collagen formation and epithelial regeneration were significantly higher in the study groups, compared to the control group (p=0.015, p=0.022, p=0.026 and p=0.002, p=0.001, p=0.002). Conclusion: Both basic fibroblast growth factor and adipose tissuederived mesenchymal stem cells increased epithelial regeneration and connective tissue organization in this rat model. They may be used as an adjuvant therapy to surgical resection in patients undergoing tracheal resection.

Interleukin-1 superfamily member, interleukin-33, in periodontal diseases

Interleukin (IL) -33 is a nuclear protein that is released from damaged cells and acts as an alarmin. We investigated the expression of IL-33 in human gingival fibroblasts after stimulation by tumor necrosis factor alpha (TNF-α). Human periodontal tissue samples were collected and fixed in phosphate-buffered 4% formalin in saline and processed to paraffin blocks. TNF-α was immunostained in samples of ten periodontitis patients and ten controls. Human gingival fibroblasts were isolated using an explant culture technique. The influence of TNF-α on IL-33 in gingival fibroblasts was analyzed using enzyme-linked immunosorbent assay (ELISA). The number of TNF-α positive cells was significantly greater in periodontitis samples than in controls. TNF-α was located mainly in macrophage- and fibroblast-like cells, vascular endothelial cells and epithelial cells. Analysis of IL-33 expression in cell culture lysates showed that TNF-α induced IL-33 in cultured gingival fibroblasts. Periodontitis samples are characterized by Th2 cell dominance, which has been linked to anti-inflammatory responses and periodontal repair. TNF-α-induced IL-33 may link inflammation directly to the IL-33-dependent stimulation of Th2 cytokine producing cells and participate in the induction of lymphocytes, which results in protective, anti-inflammatory and reparative responses.

Automated Analysis of Neurite Outgrowth in Mouse Retinal Explants

Despite intensive research efforts over the past years, regeneration of injured axons in the central nervous system remains elusive. In the quest for neurostimulatory agents that promote regeneration, well-defined models and analysis methods are required. Tissue explant cultures closely resemble the in vivo situation, making them ideal to study the effect of compounds on the neuro-glial network. This study reports the optimization of an explant culture technique using retinas of neonatal mice and the development of an analysis script that allows for rapid and automated analysis of neurite outgrowth from these explants. The key features of this script (i.e., local thresholding and form selection) allow for swift and unbiased detection of neurite outgrowth. The novel analysis method is compared with two commonly used manual methods and successfully validated by performing dose-response studies with molecules known to either inhibit (anti–β1-integrin antibody) or stimulate (brain-derived neurotrophic factor and ciliary neurotrophic factor) neurite outgrowth from retinal explants. Finally, the new analysis script is used to study whether retinal explant origin has any effect on neurite outgrowth.

Protocol: Ex Vivo Culture of Mouse Embryonic Mammary Buds

The explant culture techniques of embryonic tissues allow continuous monitoring of organ growth and morphogenesis ex vivo. The effect of growth factors and other soluble molecules can be examined by applying them to the culture medium. Relatively few studies have reported application of tissue culture techniques to analysis of embryonic mammary glands. Here we describe a protocol for murine mammary rudiments that permits ex vivo development up to branching stage.

Study on characteristics of in vitro culture and intracellular transduction of exogenous proteins in fibroblast cell line of Liaoning cashmere goat

Establishment of fibroblast cell lines of endangered goat breeds and research on the gene or protein functions based on the cells made a significant contribution to the conservation and utilization of genetic resources. In this study, a fibroblast cell line of Liaoning cashmere goat, frozen in 174 cryovials with 5 × 10(6) cells each, was successfully established from 60 goats ear marginal tissues using explant culture and cryopreservation techniques. Biological analysis of in vitro cultured cell line showed that, the cells were morphologically consistent with fibroblasts; the average viability of the cells was 94.9 % before freezing and 90.1 % after thawing; the growth process of cells was consisted of a lag phase, a logarithmic phase and a plateau phase; cell population doubling time was 65.5 h; more than 90 % of cells were diploid prior to the 6th generation; Neither microbial contamination nor cross-contamination was detected. To determine cell permeability, intracellular path and stability of exogenous proteins during the transduction, a TAT protein transduction domain was fused to the C-terminus of enhanced green fluorescent protein, the established fibroblast cell line was treated with the purified exogenous proteins at various concentrations by adding them to the cell culture media for 1-24 h and assayed cell morphology and protein presence, it was found that the purified exogenous proteins readily entered cells at a concentration of 0.1 mg/ml within 1.5 h and some of them could translocate into nucleus, moreover, the exogenous proteins appeared to be stable inside cells for up to 24 h.

Clinical outcomes of xeno-free allogeneic cultivated limbal epithelial transplantation for bilateral limbal stem cell deficiency

PurposeTo report the clinical outcomes of allogeneic cell-based therapy for bilateral corneal blindness due to limbal stem cell deficiency (LSCD).MethodsThis retrospective study included 28 eyes of 21 patients, at least...

Technical Advance: Decreased helper T cells and increased natural killer cells in chronic periodontitis analyzed by a novel method for isolating resident lymphocytes

During CP, the gingival environment is primed to recruit and activate homing lymphocytes. However, detailed phenotypic and functional characterization of gingival tissue resident lymphocytes has been challenging as a result of limitations associated with available isolation methods and limited availability of human samples. This study aimed to develop a novel explant culture technique for effectively isolating human gingival lymphocytes. This technique takes advantage of the natural tendency of MNCs to migrate toward a chemokine gradient generated by the gingival fibroblasts. The explant system allowed isolation of MNCs with ∼95× higher yield relative to conventional approaches. The MNC yield correlates directly with wet weights of the tissues, and maximal MNCs are isolated during the 2nd day of the culture. The explant culture method and conventional approach produce similar MNC subpopulations such as Th, Tc, and B cells. Chemokines associated with MNC migration but not cytokines associated with MNC proliferation and differentiation were produced in the explant culture. Moreover, MNC migration in response to the secreted chemokines was inhibited by PTX. T cells did not undergo proliferation during the culture. However, the isolated T cells responded to mitogenic stimulation ex vivo. A statistically, significantly decreased Th cell with reduced CD25 expression along with increased NK and NKT cells in CP are shown. However, the number of naïve NK cells was decreased significantly in MNCs, suggesting activation of NK cells in CP.

Interleukin-17A Increases Neurite Outgrowth from Adult Postganglionic Sympathetic Neurons

Inflammation can profoundly alter the structure and function of the nervous system. Interleukin (IL)-17 has been implicated in the pathogenesis of several inflammatory diseases associated with nervous system plasticity. However, the effects of IL-17 on the nervous system remain unexplored. Cell and explant culture techniques, immunohistochemistry, electrophysiology, and Ca2+ imaging were used to examine the impact of IL-17 on adult mouse sympathetic neurons. Receptors for IL-17 were present on postganglionic neurons from superior mesenteric ganglia (SMG). Supernatant from activated splenic T lymphocytes, which was abundant in IL-17, dramatically enhanced axonal length of SMG neurons. Importantly, IL-17-neutralizing antiserum abrogated the neurotrophic effect of splenocyte supernatant, and incubation of SMG neurons in IL-17 (1 ng/ml) significantly potentiated neurite outgrowth. The neurotrophic effect of IL-17 was accompanied by inhibition of voltage-dependent Ca2+ influx and was recapitulated by incubation of neurons in a blocker of N-type Ca2+ channels (ω-conotoxin GVIA; 30 nM). IL-17-induced neurite outgrowth in vitro appeared to be independent of glia, as treatment with a glial toxin (AraC; 5 μM) did not affect the outgrowth response to IL-17. Moreover, application of the cytokine to distal axons devoid of glial processes enhanced neurite extension. An inhibitor of the NF-κB pathway (SC-514; 20 μM) blocked the effects of IL-17. These data represent the first evidence that IL-17 can act on sympathetic somata and distal neurites to enhance neurite outgrowth, and identify a novel potential role for IL-17 in the neuroanatomical plasticity that accompanies inflammation.

Clinical Outcomes of Repeat Autologous Cultivated Limbal Epithelial Transplantation for Ocular Surface Burns

To report the clinical outcomes of repeat autologous cultivated limbal epithelial transplantation in patients with recurrence of limbal stem cell deficiency after a failed primary procedure. Retrospective case series. This study included 50 patients, above 8 years of age, with clinically diagnosed unilateral limbal stem cell deficiency following ocular surface burns, treated between 2001 and 2010. Following failure of primary surgery all patients underwent a repeat limbal biopsy from the unaffected eye. The limbal cells were expanded ex vivo on a human amniotic membrane substrate for 10 to 14 days using a completely xeno-free explant culture technique. The resulting cultured epithelial monolayer and amniotic membrane were transplanted onto the patient's affected eye. All patients underwent a comprehensive ophthalmic examination of both eyes at every follow-up visit. Postoperative corneal surface stability, change in visual acuity, and complications were objectively analyzed. At a mean follow-up of 2.3±1.4 (median: 1.96, range: 1 to 7.5) years, 33 of the 50 recipient eyes (66%) maintained a completely epithelialized, avascular, and clinically stable corneal surface. A 2-line improvement in visual acuity was seen in 38 of the 50 recipient eyes (76%). None of the donor eyes developed any clinical features of ocular surface disease, conjunctival overgrowth of the donor site, or decrease in vision throughout the follow-up period. Repeat autologous cultivated limbal epithelial transplantation successfully restores corneal epithelial stability and improves vision in eyes with recurrence of limbal stem cell deficiency, following failed primary surgery for ocular burns, without adversely affecting donor eyes.

Isolation, culturing and characterization of rat adipose tissue-derived mesenchymal stem cells: a simple technique

In this study, our aim was to develop a new simple technique for isolation of mesenchymal stem cells from adipose tissue. For this purpose, mesenchymal stem cells were isolated from rat adipose tissue by using the primary explant culture technique. When the cells became confluent, they were passaged 4 times by using the standard trypsinization method with trypsin/EDTA solution. Cells at second passage were characterized by using immunofluorescence staining against CD13 and CD29 markers. The results showed that these cultured cells were positive for CD13 and CD29 markers. Flow cytometry analysis was also done against CD29, CD90, CD54, MHC Class I, CD45, CD106, and MHC Class II for characterization of mesenchymal stem cells. The results of flow cytometry analysis showed that these cells were mesenchymal stem cells. Half of the cells were cryopreserved at all passages for future applications. It is thought that these mesenchymal stem cells can be used in therapy of cardiovascular diseases as an alternative technique in the near future.

Matrix metalloproteinase expression in primary lung fibroblasts of layer type chickens

We attempted to determine the growth characteristics of cultured lung fibroblasts of layer type chickens and to investigate presence of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in these cells in vitro. Lung fibroblasts were isolated, characterized and subcultured from one-day-old layer type chicken lungs. Two different methods, explant culture and enzymatic techniques, were used for culturing and the results were compared. The presence of MMP-2 and TIMP-1 was shown in cultured fibroblasts by immunocytochemical staining, immune blotting and zymography methods. Immune expressions of neither MMP-9 nor TIMP-2 enzymes could be detected.

Clinical outcomes of xeno-free autologous cultivated limbal epithelial transplantation: a 10-year study

PurposeOcular burns can damage the corneal epithelial stem cells located at the limbus. This study evaluated the efficacy of xeno-free autologous cell-based treatment of limbal stem cell deficiency.MethodsThis retrospective study...

Esophageal smooth muscle cells dedifferentiate with loss of α-smooth muscle actin expression after 8 weeks of explant expansion in vitro culture: Implications on esophagus tissue engineering

BACKGROUND: Esophagus tissue engineering using the hybrid-construct approach involves assembly of the esophageal components to engineer the organ. This study investigated the dedifferentiation and loss in expression of esophageal smooth muscle after explant expansion in culture. METHODS: Ovine esophagus smooth muscle cells (OESMC) were sourced from adult biopsies and expanded using the explant culture technique. The explants were maintained under static in vitro tissue culture. Flow cytometry analysis was performed for α-smooth muscle actin (α-SMA) expression at intervals of 4 weeks for up to 8 weeks. RESULTS: After 4 weeks 58.5% OESMC exhibited α-SMA which decreased to 28.5% after 6 weeks in culture. After 8 weeks in culture a mere 1.3% OESMC demonstrated α-SMA expression. CONCLUSIONS: OESMC proliferated using the explant technique exhibit a total loss of α-SMA expression after 8 weeks of static in vitro culture. This information is crucial for tissue engineering of the esophagus using the hybrid-construct approach.