Development of novel strategies for the isolation of piglet testis cells with a high proportion of gonocytes

2010 ◽  
Vol 22 (7) ◽  
pp. 1057 ◽  
Author(s):  
Yanfei Yang ◽  
Mehran Yarahmadi ◽  
Ali Honaramooz
Keyword(s):  
In Vitro Study ◽  
Enzymatic Digestion ◽  
Cell Isolation ◽  
Live Cells ◽  
Germline Stem Cell ◽  
Isolated Cells ◽  
Cell Potential ◽  
Mechanical Methods ◽  
Neonatal Testis

Gonocytes have germline stem cell potential and are present in the neonatal testis, comprising 5–10% of freshly isolated testis cells. Maximising the number and proportion of gonocytes among freshly isolated testis cells will greatly facilitate their subsequent purification and in vitro study and manipulation. Seven experiments were conducted to evaluate the effects of multiple factors on the efficiency of testis cell isolation from neonatal pigs. We found that the use of a lysis buffer led to elimination of erythrocytes without adversely affecting testis cell isolation. Approximately ninefold as many live cells could be harvested by enzymatic digestion of testis tissues compared with mechanical methods. Digestion with collagenase–hyaluronidase–DNase followed by trypsin resulted in the highest recovery of live cells. However, the proportion of gonocytes (∼7%) did not differ between the mechanical and enzymatic methods of testis cell isolation. Pretreatment of the tissue with cold enzymes increased the recovery of live testis cells. New strategies of combining a gentle enzymatic digestion with two rounds of vortexing resulted in the isolation of testis cells with very high gonocyte proportion. The efficiency of these novel methods could be further optimised to collect testis cells with a gonocyte proportion of approximately 40%. This novel three-step testis cell isolation strategy can be completed within 1 h and can harvest approximately 17 × 106 live gonocytes per g testis tissue. Therefore, in addition to elucidating the effects of several factors on testis cell isolation, we developed a novel strategy for the isolation of testis cells that yielded approximately 40% gonocytes in the freshly isolated cells (i.e. four- to eight-fold higher than the proportions obtained using current strategies). This strategy has instant applications in the purification of gonocytes.

Effects of medium and hypothermic temperatures on preservation of isolated porcine testis cells

2010 ◽  
Vol 22 (3) ◽  
pp. 523 ◽  
Author(s):  
Yanfei Yang ◽  
Ali Honaramooz
Keyword(s):  
Trypan Blue ◽  
Cell Isolation ◽  
Live Cell ◽  
Live Cells ◽  
Cell Recovery ◽  
Trypan Blue Exclusion ◽  
Culture Studies ◽  
Porcine Testis ◽  
Peritubular Cells

The effects of medium and hypothermic temperatures on testis cells were investigated to develop a strategy for their short-term preservation. Testes from 1-week-old piglets were enzymatically dissociated for cell isolation. In Experiment 1, testis cells were stored at either room (RT) or refrigeration (RG) temperature for 6 days in one of 13 different media. Live cell recovery was assayed daily using trypan blue exclusion. In Experiment 2, three media at RG were selected for immunocytochemical and in vitro culture studies. Live cell recovery was also assayed daily for 6 days using both trypan blue exclusion and a fluorochrome assay kit. For all media tested, significantly or numerically more live cells were maintained at RG than RT. On preservation Day 3 at RG (cell isolation day as Day 0), 20% FBS-Leibovitz resulted in the highest live cell recovery (89.5 ± 1.7%) and DPBS in the lowest (60.3 ± 1.9%). On Day 6 at RG, 20% FBS- Leibovitz also resulted in the best preservation efficiency with 80.9 ± 1.8% of Day 0 live cells recovered. There was no difference in live cell recovery detected by the two viability assays. After preservation, the proportion of gonocytes did not change, whereas that of Sertoli and peritubular cells increased and decreased, respectively. After 6 days of hypothermic preservation, testis cells showed similar culture potential to fresh cells. These results show that testis cells can be preserved for 6 days under hypothermic conditions with a live cell recovery of more than 80% and after-storage viability of 88%.

scholarly journals Collagen matrices as an improved model for in vitro study of live cells using Raman microspectroscopy

2011 ◽  
Author(s):  
F. Bonnier ◽  
P. Knief ◽  
A. D. Meade ◽  
J. Dorney ◽  
K. Bhattacharya ◽  
...  
Keyword(s):  
In Vitro Study ◽  
Raman Microspectroscopy ◽  
Vitro Study ◽  
Live Cells ◽  
Collagen Matrices ◽  
Improved Model

Isolation and characterization of the lower portion of the thin limb of Henle in primary culture

1998 ◽  
Vol 274 (4) ◽  
pp. F775-F782 ◽  
Author(s):  
Clemens Grupp ◽  
Michael Begher ◽  
David Cohen ◽  
Michael Raghunath ◽  
Hans-Eduard Franz ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Water Channel ◽  
Cell Types ◽  
Enzymatic Digestion ◽  
Lower Portion ◽  
Isolated Cells ◽  
Hormonal Stimulation ◽  
Isolation And Characterization ◽  
Thin Limb

To further characterize cells of the lower portion of the thin limb of Henle (TLHlp) under defined conditions in vitro, we developed a technique to enrich this cell population in suspension. TLHlp cells were isolated by enzymatic digestion of rat inner medulla, elimination of collecting ducts by lectin-coated beads, and differential centrifugation. Immunohistochemical staining of primary cultures of TLHlp cells with various markers revealed the preparations to be >90% pure. The hormonal stimulation pattern of PGE2 and cAMP production by arginine vasopressin, angiotensin II, and dopamine in the isolated cells also argued against significant contamination by other cell types. Staining with an antibody against the aquaporin-1 water channel showed the distribution of cells from the ascending and descending limbs to be approximately equal in the isolated population. This technique allows the enrichment of cells from the lower portion of the thin limb of Henle in suspension to a very high degree of purity with the option to start primary cultures. Because these segments of the tubular system in particular are relatively inaccessible for microdissection, the presented method renders the possibility of addressing new questions regarding these tubular segments under defined conditions in vitro.

A comparison of cytokine and hormone production by decidual cells and tissue explants

1996 ◽  
Vol 151 (2) ◽  
pp. 309-313 ◽  
Author(s):  
L B Lonsdale ◽  
M G Elder ◽  
M H F Sullivan
Keyword(s):  
Transforming Growth Factor ◽  
Placental Tissue ◽  
Enzymatic Digestion ◽  
Isolated Cells ◽  
Hormone Production ◽  
General Process ◽  
Decidual Cells ◽  
Cells Cultured

Abstract Previous work has shown that enzymatic digestion of human placental tissue can induce the production of the cytokine interleukin-1β. Most studies of the feto-maternal interface of human pregnancy have used decidual cells prepared in a similar way, but the effects of tissue dissociation on the production of growth factors, cytokines, prostaglandins or hormones have not been investigated. Our studies show human decidual explants produce substantially lower levels of a range of factors than do human decidual cells cultured under the same conditions, indicating that induction may be a general process during the dissociation of tissues in vitro as the production of interleukins-1β, -6 and -8, granulocyte-macrophage colony-stimulating factor, transforming growth factor-β2, tissue necrosis factor-α, prostaglandins E2 and F2α, and prolactin were all affected. The induction of cytokine production (expressed per mg tissue protein) ranged from 10- to 300-fold, indicating that isolated cells cultured in vitro may not reflect accurately the in vivo situation. Journal of Endocrinology (1996) 151, 309–313

scholarly journals Minced Umbilical Cord Fragments as a Source of Cells for Orthopaedic Tissue Engineering: An In Vitro Study

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
A. Marmotti ◽  
S. Mattia ◽  
M. Bruzzone ◽  
S. Buttiglieri ◽  
A. Risso ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Bone Marrow ◽  
Stem Cell ◽  
Umbilical Cord ◽  
In Vitro Study ◽  
Enzymatic Digestion ◽  
Mean Value ◽  
Simple Protocol ◽  
Multilineage Potential

A promising approach for musculoskeletal repair and regeneration is mesenchymal-stem-cell- (MSC-)based tissue engineering. The aim of the study was to apply a simple protocol based on mincing the umbilical cord (UC), without removing any blood vessels or using any enzymatic digestion, to rapidly obtain an adequate number of multipotent UC-MSCs. We obtained, at passage 1 (P1), a mean value of4,2×106cells (SD 0,4) from each UC. At immunophenotypic characterization, cells were positive for CD73, CD90, CD105, CD44, CD29, and HLA-I and negative for CD34 and HLA-class II, with a subpopulation negative for both HLA-I and HLA-II. Newborn origin and multilineage potential toward bone, fat, cartilage, and muscle was demonstrated. Telomere length was similar to that of bone-marrow (BM) MSCs from young donors. The results suggest that simply collecting UC-MSCs at P1 from minced umbilical cord fragments allows to achieve a valuable population of cells suitable for orthopaedic tissue engineering.

scholarly journals Early Expression of Bone Matrix Proteins in Osteogenic Cell Cultures

2003 ◽  
Vol 51 (5) ◽  
pp. 633-641 ◽  
Author(s):  
Paulo Tambasco de Oliveira ◽  
Sylvia Francis Zalzal ◽  
Kazuharu Irie ◽  
Antonio Nanci
Keyword(s):  
Bone Matrix ◽  
Enzymatic Digestion ◽  
Matrix Proteins ◽  
Cell Labeling ◽  
Type I ◽  
Isolated Cells ◽  
Osteoblast Activity ◽  
Early Expression ◽  
Almost All

Osteogenic cells express some matrix proteins at early culture intervals. The aim of this study was to determine if, and in what proportion, cells used for plating contain bone sialoprotein (BSP) and osteopontin (OPN), two matrix proteins associated with initial events in bone formation. Their pattern of expression, as well as that of fibronectin (FN) and type I pro-collagen, was also examined at 6 hr and at 1 and 3 days. The cells were obtained by enzymatic digestion of newborn rat calvariae, and grown on glass coverslips. Cytocentrifuge preparations of isolated cells and coverslips were processed for single or dual immunolabeling with monoclonal and/or polyclonal primary antibodies, followed by fluorochrome-conjugated antibodies. The cell labeling was mainly associated with perinuclear elements. OPN was also distinctively found at peripheral cytoplasmic sites. About 31% of isolated cells were OPN-positive and 18% were BSP-positive. After 1 day, almost 50% of cells were immunoreactive for OPN and for type I pro-collagen, and still less than 20% reacted for BSP. Approximately 7% exhibited peripheral staining for OPN. Almost all cells were associated with extracellular FN. However, only 15% showed intracellular labeling. These results indicate that an important proportion of cells used for plating contain BSP and OPN, a situation that should be taken into consideration in experimental analyses of osteoblast activity in vitro.

scholarly journals Endonuclease increases efficiency of osteoblast isolation from murine calvariae

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yosuke Asano ◽  
Yoshinori Matsumoto ◽  
Jose La Rose ◽  
Fang He ◽  
Takayuki Katsuyama ◽  
...  
Keyword(s):  
Bone Resorption ◽  
In Vitro Study ◽  
Genetically Engineered ◽  
Vitro Study ◽  
In Vitro Experiments ◽  
Isolated Cells ◽  
Cellular Identity ◽  
And Function ◽  
Number Of Cells

AbstractBone is a highly dynamic organ that undergoes remodeling equally regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. To clarify the regulation of osteoblastogenesis, primary murine osteoblasts are required for an in vitro study. Primary osteoblasts are isolated from neonatal calvariae through digestion with collagenase. However, the number of cells collected from one pup is not sufficient for further in vitro experiments, leading to an increase in the use of euthanized pups. We hypothesized that the viscosity of digested calvariae and digestion solution supplemented with collagenase results in cell clumping and reduction of isolated cells from bones. We simply added Benzonase, a genetically engineered endonuclease that shears all forms of DNAs/RNAs, in order to reduce nucleic acid-mediated viscosity. We found that addition of Benzonase increased the number of collected osteoblasts by three fold compared to that without Benzonase through reduction of viscosity. Additionally, Benzonase has no effect on cellular identity and function. The new osteoblast isolation protocol with Benzonase minimizes the number of neonatal pups required for an in vitro study and expands the concept that isolation of other populations of cells including osteocytes that are difficult to be purified could be modified by Benzonase.

VARYING RESPONSE TO LUTEINIZING HORMONE OF TWO LUTEAL CELL TYPES ISOLATED FROM BOVINE CORPUS LUTEUM

1979 ◽  
Vol 83 (3) ◽  
pp. 303-NP ◽  
Author(s):  
JOCELYNE URSELY ◽  
PIERRE LEYMARIE
Keyword(s):  
Corpus Luteum ◽  
In Vitro Study ◽  
Average Diameter ◽  
Cell Types ◽  
Enzymatic Digestion ◽  
Cell Suspensions ◽  
Small Cells ◽  
Luteal Cell

Luteal cell suspensions obtained by enzymatic digestion of pregnant cow corpus luteum were found to be heterogenous and mainly made up of two types of cells of different sizes. The large cells (37 μm, average diameter) could be separated from the small ones (18 μm, average diameter) by sedimentation at unit gravity in a gradient of Ficoll–bovine serum albumin. A comparative in-vitro study of the synthesis of progesterone by the two types of cells indicated striking differences between them. The average content and the synthesis of progesterone in the absence and presence of a saturating dose of bovine LH after incubation for 2 h were 0·07, 0·12 and 6·9 pg/cell for the small cells and 0·65, 2 and 10 pg/cell for the large ones. Moreover, the sensitivity to low concentrations of LH was 100 to 1000 times higher for the small cells than for the large ones. oestradiol-17β at concentrations ranging from 5 × 10−10 to 5 × 10−4 mol/l exerted a dose–dependent inhibition on the stimulation of LH in both cell types. These results suggest a possible involvement of both cell types in the synthesis of progesterone in vivo with a greater contribution by the small cells to stimulation induced by LH. Moreover, it appears that small cell suspensions could be a useful model system for in-vitro studies of the control of the synthesis of progesterone in cow corpus luteum.

scholarly journals The Effect of Enzymatic Digestion on Cultured Epithelial Autografts

2019 ◽  
Vol 28 (5) ◽  
pp. 638-644 ◽  
Author(s):  
M. Skog ◽  
Petter Sivlér ◽  
Ingrid Steinvall ◽  
Daniel Aili ◽  
Folke Sjöberg ◽  
...  
Keyword(s):  
Enzymatic Digestion ◽  
Stem Cell Markers ◽  
Digestion Time ◽  
Isolated Cells ◽  
Skin Cells ◽  
Severe Burns ◽  
Significant Difference ◽  
Cultured Epithelial Autografts ◽  
The Impact

Severe burns are often treated by means of autologous skin grafts, preferably following early excision of the burnt tissue. In the case of, for example, a large surface trauma, autologous skin cells can be expanded in vitro prior to transplantation to facilitate the treatment when insufficient uninjured skin is a limitation. In this study we have analyzed the impact of the enzyme (trypsin or accutase) used for cell dissociation and the incubation time on cell viability and expansion potential, as well as expression of cell surface markers indicative of stemness. Skin was collected from five individuals undergoing abdominal reduction surgery and the epidermal compartment was digested in either trypsin or accutase. Trypsin generally generated more cells than accutase and with higher viability; however, after 7 days of subsequent culture, accutase-digested samples tended to have a higher cell count than trypsin, although the differences were not significant. No significant difference was found between the enzymes in median fluorescence intensity of the analyzed stem cell markers; however, accutase digestion generated significantly higher levels of CD117- and CD49f-positive cells, but only in the 5 h digestion group. In conclusion, digestion time appeared to affect the isolated cells more than the choice of enzyme.

scholarly journals The Benefit of Minced Cartilage Over Isolated Chondrocytes in Atelocollagen Gel on Chondrocyte Proliferation and Migration

Cartilage ◽  
2018 ◽  
Vol 12 (1) ◽  
pp. 93-101 ◽  
Author(s):  
Yusuke Tsuyuguchi ◽  
Tomoyuki Nakasa ◽  
Masakazu Ishikawa ◽  
Shigeru Miyaki ◽  
Ryosuke Matsushita ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Migration ◽  
In Vitro Study ◽  
Enzymatic Digestion ◽  
Cartilage Defects ◽  
Control Group ◽  
Chondrocyte Implantation ◽  
Matrix Production ◽  
And Migration

Objective Autologous chondrocyte implantation is a necessary procedure for the repair of articular cartilage defects; however, isolated chondrocyte implantation requires a 2-step procedure (for harvesting and implantation) and is limited by cytotoxicity due to enzymatic digestion. Therefore, in this in vitro study, we evaluated the possible benefit of using minced cartilage embedded in a 3-dimensional culture scaffold and fixed with fibrin glue, in comparison with isolated chondrocytes in atelocollagen, to induce cell migration, proliferation, and matrix production, using cartilage from patients with knee joint osteoarthritis. Design Cartilage fragments were obtained from 7 female patients with knee osteoarthritis (OA) and embedded in atelocollagen gels. As a control, chondrocytes were isolated and embedded in gels in the same manner. These composites were cultured for 3 weeks, and cell proliferation and matrix production were evaluated using histology and immunochemistry. Results Histologically, minced cartilage showed cell migration from the cartilage fragments into the gel, with the Bern score and cell count in the minced cartilage group being significantly higher than those in the control group. Immunohistochemistry revealed that the number of Ki67-positive cells, the expression of LECT-1 and TGF-β, and the glycosaminoglycan content were significantly higher in the minced cartilage than in the control group. Minced cartilage exhibited superior cell migration, proliferation, and glycosaminoglycan content than isolated chondrocytes. Conclusion Our findings support that minced cartilage has a favorable potential for cell proliferation and matrix production compared with the isolated chondrocytes after enzymatic treatment.

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