scholarly journals Optimization of Co-Culture Conditions for a Human Vascularized Adipose Tissue Model

2020 ◽  
Vol 7 (3) ◽  
pp. 114 ◽  
Author(s):  
Feipeng Yang ◽  
Ronald N. Cohen ◽  
Eric M. Brey
Keyword(s):  
Adipose Tissue ◽  
Endothelial Cell ◽  
Metabolic Diseases ◽  
Culture Media ◽  
Culture Conditions ◽  
Extensive Evaluation ◽  
Proliferation And Differentiation ◽  
And Function ◽  
Tissue Models

In vitro adipose tissue models can be used to provide insight into fundamental aspects of adipose physiology. These systems may serve as replacements for animal models, which are often poor predictors of obesity and metabolic diseases in humans. Adipose tissue consists of a rich vasculature that is essential to its function. However, the study of endothelial cell–adipocyte interactions has been challenging due to differences in culture conditions required for the survival and function of each cell type. To address this issue, we performed an extensive evaluation of the cell culture media composition to identify the conditions optimal for the co-culture of endothelial cells and adipocytes. The effects of individual media factors on cell survival, proliferation, and differentiation were systematically explored. Several media factors were determined to disrupt the co-culture system. Optimized culture conditions were identified and used to generate a vascularized human adipose microtissue. An interconnected vascular network was established within an adipose micro-tissue, and the networks were anastomosed with perfused channels to form a functional network. In conclusion, media conditions were identified that enabled endothelial cell–adipocyte co-culture and were used to support the formation of a vascularized adipose tissue within a microfluidic device.

Biophysical stimulation for in vitro engineering of functional cardiac tissues

2017 ◽  
Vol 131 (13) ◽  
pp. 1393-1404 ◽  
Author(s):  
Anastasia Korolj ◽  
Erika Yan Wang ◽  
Robert A. Civitarese ◽  
Milica Radisic
Keyword(s):  
Cardiac Tissue ◽  
Culture Media ◽  
Culture Conditions ◽  
Lineage Specification ◽  
Cardiac Tissues ◽  
Cardiac Lineage ◽  
And Function ◽  
Tissue Models

Engineering functional cardiac tissues remains an ongoing significant challenge due to the complexity of the native environment. However, our growing understanding of key parameters of the in vivo cardiac microenvironment and our ability to replicate those parameters in vitro are resulting in the development of increasingly sophisticated models of engineered cardiac tissues (ECT). This review examines some of the most relevant parameters that may be applied in culture leading to higher fidelity cardiac tissue models. These include the biochemical composition of culture media and cardiac lineage specification, co-culture conditions, electrical and mechanical stimulation, and the application of hydrogels, various biomaterials, and scaffolds. The review will also summarize some of the recent functional human tissue models that have been developed for in vivo and in vitro applications. Ultimately, the creation of sophisticated ECT that replicate native structure and function will be instrumental in advancing cell-based therapeutics and in providing advanced models for drug discovery and testing.

scholarly journals Developmental aspects of adipose tissue in GH receptor and prolactin receptor gene disrupted mice: site-specific effects upon proliferation, differentiation and hormone sensitivity

2006 ◽  
Vol 191 (1) ◽  
pp. 101-111 ◽  
Author(s):  
David J Flint ◽  
Nadine Binart ◽  
Stephanie Boumard ◽  
John J Kopchick ◽  
Paul Kelly
Keyword(s):  
Adipose Tissue ◽  
Receptor Gene ◽  
Metabolic Effects ◽  
Wild Type ◽  
Extrinsic Factors ◽  
Differentiation Potential ◽  
Site Specific ◽  
Proliferation And Differentiation

Direct metabolic effects of GH on adipose tissue are well established, but effects of prolactin (PRL) have been more controversial. Recent studies have demonstrated PRL receptors on adipocytes and effects of PRL on adipose tissue in vitro. The role of GH in adipocyte proliferation and differentiation is also controversial, since GH stimulates adipocyte differentiation in cell lines, whereas it stimulates proliferation but inhibits differentiation of adipocytes in primary cell culture. Using female gene disrupted (ko) mice, we showed that absence of PRL receptors (PRLRko) impaired development of both internal and s.c. adipose tissue, due to reduced numbers of adipocytes, an effect differing from that of reduced food intake, where cell volume is decreased. In contrast, GHRko mice exhibited major decreases in the number of internal adipocytes, whereas s.c. adipocyte numbers were increased, even though body weight was decreased by 40–50%. The changes in adipose tissue in PRLRko mice appeared to be entirely due to extrinsic factors since preadipocytes proliferated and differentiated in similar fashion to wild-type animals in vitro and their response to insulin and isoproterenol was similar to wild-type animals. This contrasted with GHRko mice, where s.c. adipocytes proliferated, differentiated, and responded to hormones in identical fashion to controls, whereas parametrial adipocytes exhibited markedly depressed proliferation and differentiation potential and failed to respond to insulin or noradrenaline. Our results provide in vivo evidence that both GH and PRL stimulate differentiation of adipocytes but that the effects of GH are site specific and induce intrinsic changes in the precursor population, which are retained in vitro.

The effect of osmolarity on mouse parthenogenesis

Development ◽  
1974 ◽  
Vol 31 (2) ◽  
pp. 513-526
Author(s):  
M. H. Kaufman ◽  
M. A. H. Surani
Keyword(s):  
Protein Synthesis ◽  
Culture Medium ◽  
Culture Media ◽  
Polar Body ◽  
Amino Acid Mixture ◽  
Culture Conditions ◽  
Follicle Cells ◽  
Acid Mixture ◽  
Second Polar Body

Eggs from (C57B1 × A2G)F1 mice were activated by treatment with hyaluronidase, which removed the follicle cells, and cultured in vitro. Observations were made 6–8 h after hyaluronidase treatment to determine the frequency of activation and the types of parthenogenones induced. Cumulus-free eggs resulting from hyaluronidase treatment were incubated for 2¼ h in culture media of various osmolarities. The frequency of activation was found to be dependent on the postovulatory age of oocytes, while the types of parthenogenones induced were dependent on the osmolarity of the in vitro culture medium and their postovulatory age. Culture in low osmolar medium suppressed the extrusion of the second polar body (2PB). This decreased the incidence of haploid eggs with a single pronucleus and 2PB and immediately cleaved eggs from 97·5% to 42·3% of the activated population. Where 2PB extrusion had been suppressed, 97·4% of parthenogenones contained two haploid pronuclei. Very few were observed with a single and presumably diploid pronucleus. Serial observations from 11 to 18 h after hyaluronidase treatment were made on populations of activated eggs as they entered the first cleavage mitosis after 2¼ h incubation in medium either of normal (0·287 osmol) or low (0·168 osmol) osmolarity. A delay in the time of entry into the first cleavage mitosis similar to the duration of incubation in low osmolar medium was observed. Further, eggs were incubated in control and low osmolar culture media containing uniformly labelled [U-14C]amino acid mixture to examine the extent of protein synthesis in recently activated eggs subjected to these culture conditions. An hypothesis is presented to explain the effect of incubation in low osmolar culture medium in delaying the first cleavage mitosis.

scholarly journals Bioreactors as physiologicallike in vitro models

2020 ◽  
Author(s):  
Sara Mantero ◽  
Federica Boschetti
Keyword(s):  
Culture Conditions ◽  
In Vitro Models ◽  
In Vivo Models ◽  
In Vitro Development ◽  
Culture Cells ◽  
Vitro Development ◽  
Tissue Models ◽  
Mechanical Stretching

Bioreactors are powerful tools for in vitro development of engineered substitutes through controlled biological, physical, and mechanical culture conditions: bioreactor technology allows a closer in vitro replication of native tissues. One of bioreactors applications is the design of in vitro 3D tissue models as a bridge between 2D and in vivo models, allowing the application of 3R (replacement, reduction, refinement) principle. To this aim, bioreactors can be used to culture cells seeded on engineered scaffolds under in vivo-like conditions. Another key use of bioreactors is for perfusion decellularization of tissues and organs to be used as scaffolds. This contribution describes a dynamic stretching. bioreactor, imposing a mechanical stretching to the cultured constructs, allowing the development of skeletal muscle engineered constructs, and a decellularization bioreactor, designed for decellularization of blood vessels.

Dexamethasone inhibits prostaglandin release from rabbit coronary microvessel endothelium

1986 ◽  
Vol 250 (6) ◽  
pp. C970-C977 ◽  
Author(s):  
R. M. Rosenbaum ◽  
C. D. Cheli ◽  
M. E. Gerritsen
Keyword(s):  
Endothelial Cell ◽  
Culture Media ◽  
Conditioned Media ◽  
Microvascular Endothelial Cell ◽  
Dexamethasone Treatment ◽  
Stimulatory Action ◽  
Prostaglandin Release ◽  
Cell Release ◽  
Microvascular Endothelial

The effects of dexamethasone on prostaglandin secretion by cultivated rabbit coronary microvascular endothelial (RCME) cells were investigated. Incubation of RCME cells with dexamethasone resulted in a time- and concentration-dependent decrease in prostaglandin accumulation in the culture media and reduced basal and A23187-stimulated prostaglandin (PG) E2 and 6-keto-PGF1 alpha release. The maximal effects of dexamethasone (50-80% inhibition) were achieved after 16-18 h of incubation with the steroid at a final concentration of 10(-7) M. The effects of dexamethasone treatment were partially reversed 24 h after removal of the steroid from the culture media. Dexamethasone treatment did not reduce arachidonic acid-stimulated prostaglandin synthesis, indicating that the level of inhibition was proximal to that of cyclooxygenase. The inhibitory effects of dexamethasone could be prevented by pretreatment of the RCME cells with actinomycin D or cycloheximide, suggesting a requirement for protein synthesis in the inhibitory action of dexamethasone. Conditioned media from dexamethasone-treated cells contained a factor that inhibited porcine pancreatic phospholipase A2 (PLA2) in vitro. Transfer of conditioned media from dexamethasone-treated cells to untreated cells did not reduce basal or stimulated prostaglandin release; in contrast, a stimulatory action was consistently observed. Adherence of rabbit peripheral polymorphonuclear leukocytes (PMN) to RCME cells was reduced when the leukocytes were pretreated with 10(-7) M dexamethasone (4 h). However, dexamethasone pretreatment of the RCME cells did not significantly effect granulocyte adhesion. Thus coronary microvascular endothelial cell prostaglandin production is regulated by glucocorticoids, and glucocorticoid-pretreated microvascular endothelial cell release an inhibitor of PLA2 activity into the culture media.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemodynamic Shear Stress And Endothelial Cell Function: In Vitro Studies

1981 ◽  
Author(s):  
M A Gimbrone ◽  
C F Dewey ◽  
P F Davies ◽  
S R Bussolari
Keyword(s):  
Shear Stress ◽  
Endothelial Cell ◽  
Fluid Shear Stress ◽  
Cell Structure ◽  
Structure And Function ◽  
Cellular Migration ◽  
Shear Stresses ◽  
Endothelial Cell Function ◽  
And Function

The vascular endothelial lining in vivo is constantly subjected to hemodynamic shear stresses resulting from normal and altered patterns of blood flow. To facilitate the study of effects of fluid shear stress on endothelial cell structure and function, we have developed an in vitro system, utilizing a cone-plate apparatus, to subject coverslip cultures of bovine aortic endothelial cells (BAEC) to controlled levels of shear (up to 102 dynes/cm2) in either laminar or turbulent flow. The magnitude and direction of shear stress within the system are accurately known from both theory and experimental measurements. The data reported here are for laminar flow. Subconfluent BAEC cultures continuously exposed to 1-5 dynes/cm2 shear proliferated at a rate comparable to that of static cultures, and postconfluent monolayers appeared unaltered morphologically for up to 1 week. In contrast, BAEC cultures (both postconfluent and subconfluent) exposed to 8 dynes/cm2 developed dramatic, time-dependent morphological changes. By 48 hrs, cells uniformly assumed an ellipsoidal configuration, with their major axes aligned in the direction of flow. Exposure to >10 dynes/cm2 caused variable cell detachment from plain glass substrates. Cellular migration into linear “wounds”, created in confluent areas, was influenced by both the direction and amplitude of applied shear. Exposure to 8 dynes/ cm2 induced functional alterations, including increased fluid (bulk phase) endocytosis, prostaglandin production and platelet reactivity. These observations indicate that fluid mechanical forces can directly influence endothelial cell structure and function. Hemodynamic modulation of endothelial cell behavior may be relevant to normal vessel wall physiology, as well as the pathogenesis of atherosclerosis and thrombosis.

Influence of somatotropin on lipid metabolism and IGF gene expression in porcine adipose tissue

1992 ◽  
Vol 263 (4) ◽  
pp. E637-E645 ◽  
Author(s):  
C. K. Wolverton ◽  
M. J. Azain ◽  
J. Y. Duffy ◽  
M. E. White ◽  
T. G. Ramsay
Keyword(s):  
Gene Expression ◽  
Adipose Tissue ◽  
Dietary Fat ◽  
Subcutaneous Adipose Tissue ◽  
Proliferation And Differentiation ◽  
Igf I ◽  
Systemic Factors ◽  
Subcutaneous Adipose ◽  
Acid Esterification

The present study was designed to evaluate the effects of porcine somatotropin (pST) treatment (2 mg/day) and dietary fat (10%) separately and in combination on the metabolic activity of subcutaneous adipose tissue, serum adipogenic activity, and insulin-like growth factor (IGF) gene expression within adipose tissue from growing 5- to 6-mo-old barrows. This study attempted to determine how these factors might contribute to the reported changes in adiposity of treated swine. Biopsies of adipose tissue were collected after 28 days of treatment following anesthesia with thiopental sodium (15 mg/kg iv). Somatotropin inhibited in vitro glucose oxidation and lipogenesis in adipose tissue but did not affect fatty acid esterification. Adipogenic activity of serum was not altered by pST treatment. Subcutaneous adipose tissue contained mRNA for IGF-I and -II, and pST administration increased the abundance of IGF-I mRNA. Dietary fat had no effect on these variables. Thus somatotropin reduces glucose metabolism in porcine subcutaneous adipose tissue. Preadipocyte proliferation and differentiation are not affected by somatotropin through its actions on systemic factors. Dietary fat provides no additional benefit in combination with pST administration to affect accretion of adipose tissue in growing swine.

187 INCREASED NUMBER OF EXPANDED BOVINE BLASTOCYSTS IN SOF AND CR1 RELATIVE TO KSOM

2007 ◽  
Vol 19 (1) ◽  
pp. 210
Author(s):  
D. M. Kohl ◽  
R. L. Monson ◽  
L. E. Enwall ◽  
J. J. Rutledge
Keyword(s):  
Gene Expression ◽  
Culture Media ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Culture Conditions ◽  
Pregnancy Rates ◽  
Embryo Morphology ◽  
Bovine Embryos ◽  
Chi Square Analysis

Assessment of morphological stage grade is a subjective procedure. Stage grade is of vital importance to, among other things, recipient synchrony for the purpose of establishing successful pregnancies. Asynchronous embryo transfer has led to decreases in pregnancy rates (Farin et al. 1995 Biol. Reprod. 52, 676–682) and has been implicated in contributing to large offspring syndrome (Young et al. 1996 Theriogenology 45, 231). Differences in embryo kinetics based on culture conditions have been well documented (Mello et al. 2005 Reprod. Fert. Dev. 17, 221 abst). Whether such differences are the result of species, breed, metabolic stress, sire effects, or separation from an in vivo environment has yet to be determined. The correlation between oxygen respiration rates and embryo morphology as well as embryo diameter in bovine embryos produced in vitro has shown promise in the development of a more objective predictor of embryo quality and perhaps pregnancy initiation (Lopes et al. 2005 Reprod. Fert. Dev. 17, 151 abst). As well, recent examination of gene expression patterns of in vitro-derived bovine embryos seems to indicate that longer periods of in vitro culture are associated with lower rates of embryo survival (Lonergan et al. 2006 Theriogenology 65, 137–152). We hypothesize that differences do exist in the number, rate, and morphological appearance of blastocysts and that these parameters are in large part based on culture conditions in vitro. The objective of this experiment was to determine the timing and distribution of blastocyst formation of in vitro-produced bovine embryos cultured in SOF8, CR18AA, and KSOM8, under a standard incubation environment. Bovine ovaries from a local abattoir were aspirated and matured for 18-22. Oocytes were fertilized with frozen-thawed Percoll-separated semen from a Holstein bull. Presumptive zygotes were vortexed to remove cumulus cells and placed into 3 different culture media in a highly humidified atmosphere containing 20% oxygen, 5% carbon dioxide, and compressed air at 38.5�C. Embryos were evaluated specifically at 168 h post-insemination (Day 7) and assigned a morphological stage grade (IETS) to determine fixed time point differences. A total of 6 complete replicates were performed. Only embryos exhibiting the presence of a blastocoel at this time were documented (early blast, mid-blast, expanded blast). At 168 h post-insemination, there were no significant differences in the total number of embryos reaching early or mid-blast stage in any of the media. However, chi-square analysis revealed an increase in the number of expanded blastocysts in SOF (n = 813) and CR1 (n = 838) treatments compared to KSOM (n = 824; P < 0.0001). Expanded blastocysts in SOF were also greater in number than in CR1 (P < 0.05). Embryo selection based on development to the expanded blastocyst stage on Day 7 may prove useful in increasing pregnancy rates, and may validate qualitative correlations based on oxygen consumption and gene expression profiles for embryos produced in vitro.

Effect of culture conditions on artificial activation of porcine oocytes matured in vitro

1996 ◽  
Vol 8 (8) ◽  
pp. 1153 ◽  
Author(s):  
N Yamauchi ◽  
H Sasada ◽  
S Sugawara ◽  
T Nagai
Keyword(s):  
In Vitro Maturation ◽  
Fetal Calf Serum ◽  
Culture Media ◽  
Calf Serum ◽  
Treated Group ◽  
Culture Conditions ◽  
Culture Period ◽  
Subsequent Response ◽  
Artificial Activation

The effects of culture media used and culture period for in vitro maturation of porcine oocytes on their subsequent response to chemical and electrical activation, were investigated. Activated oocytes were identified by the presence of a pronucleus(ei) or cleavage. Porcine oocytes were cultured for 24, 30, 36, 42 and 48 h in TCM199 with Earle's salts (199) supplemented with 10% fetal calf serum (199-FCS) before electrical stimulation. Although few oocytes were activated after 24 h and 30 h of culture (5.4% and 6.1% respectively), the percentage of activated oocytes increased significantly to 93.2% after 42 h in culture (P < 0.05); however, when the culture period was extended to 48 h, there was a significant decrease to 56.7% (P < 0.05). Oocytes were also cultured in four types of media: (1) 199-FCS; (2) 199 supplemented with 5 mg mL-1 bovine serum albumin (199-BSA); (3) Kreb's-Ringer bicarbonate solution supplemented with 10% FCS (KRB-FCS); and (4) KRB supplemented with BSA (KRB-BSA). After 42 h of culture in each medium, the oocytes were electrically activated. Although rates of maturation of oocytes cultured in the four media were similar (74.0-80.8%), all oocytes except those cultured in 199-FCS failed to be activated. In addition, oocytes were cultured for 36, 42 and 48 h in 199-FCS and then stimulated by treatment with ethanol. Significantly fewer oocytes were activated in the chemically-treated group than in the electrically-treated group. These results indicate that culture conditions used for the culture of porcine oocytes in vitro are important with respect to their subsequent response to artificial activation.

scholarly journals In vitro myelopoiesis stimulated by rapid medium exchange and supplementation with hematopoietic growth factors

Blood ◽  
1991 ◽  
Vol 78 (12) ◽  
pp. 3155-3161 ◽  
Author(s):  
RM Schwartz ◽  
SG Emerson ◽  
MF Clarke ◽  
BO Palsson
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Culture Medium ◽  
Culture Conditions ◽  
Hematopoietic Growth Factors ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Medium Exchange ◽  
Proliferation And Differentiation

Abstract We studied the effect of the combination of rapid culture medium exchange with the addition of the human hematopoietic growth factors interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (Epo) on the proliferation and differentiation of human long-term bone marrow cultures (LTBMCs). Individually and in combinations, IL-3, GM-CSF, and Epo were added to the culture medium of LTBMCs that were maintained with 50% medium volume exchange per day. The combination of IL-3 + GM-CSF + Epo generated the most prolific cultures with an order of magnitude increase in nonadherent cell production from weeks 2 through 8 in culture as compared with unsupplemented controls. Under these conditions, the cultures produced as many cells as were inoculated every 2 weeks and led to a greater than 2.5-fold expansion in terms of the number of nonadherent cells produced over a 6- to 8-week period. Furthermore, the LTBMCs produced nonadherent colony-forming unit-GM (CFU-GM) for more than 20 weeks. The rapid medium exchange combined with the addition of human hematopoietic CSFs significantly enhances the proliferation and differentiation of LTBMCs. These results indicate that addition of combinations of hematopoietic CSFs, together with a rapid medium exchange rate, can provide culture conditions that are suitable for the expansion of the progenitor cell pool and perhaps for the increased survival of hematopoietic stem cells in culture. Although these culture conditions still fall short of full reconstitution of functional human bone marrow, they provide an improved approach to hematopoietic cell culture that may permit the expansion and manipulation of progenitor cells in vitro.

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