scholarly journals To Serum or Not to Serum: Reduced-serum Based Approaches for Contact-based Co-culture of Fibroblasts and Keratinocytes for Wound Bed Studies

2020 ◽  
Author(s):  
Snehal Kadam ◽  
Madhusoodhanan Vandana ◽  
Karishma Kaushik
Keyword(s):  
Wound Closure ◽  
Culture Media ◽  
Cell Types ◽  
High Serum ◽  
Dermal Fibroblasts ◽  
Epidermal Keratinocytes ◽  
Serum Concentrations ◽  
Human Epidermal Keratinocytes ◽  
Cell Culture Media ◽  
Structure And Functions

Contact-based co-culture of fibroblasts and keratinocytes is important to study the structure and functions of the wound bed. Co-culture of these two cell types in direct contact with each other has been challenging, requiring high serum concentrations (up to 10%), feeder systems and a range of supplemental factors. These approaches are not only technically demanding, but also present scientific, cost and ethical limitations associated with high-serum concentrations. We have developed two reduced-serum approaches (1-2%) to support contact-based co-culture of human dermal fibroblasts (HDFa) and human epidermal keratinocytes (HaCaT). The two approaches include (1) Specialized cell culture media for each cell type mixed in a 1:1 ratio (KGM+FGM), and (2) Minimal media supplemented with cell-specific growth factors (MEM+GF). Co-culture could be successfully achieved by co-seeding (two cell types were introduced simultaneously), or in a layered fashion (keratinocytes seeded on top of confluent fibroblasts). With wound scratch assays, the co-cultured platforms could demonstrate cell proliferation, migration and wound closure. The reduced-serum conditions developed are simple, easy to formulate and adopt, and based on commonly-available media components. These contact-based co-culture approaches can be leveraged for wound and skin studies, and tissue bioengineering applications, potentially reducing concerns with high-serum formulations.

scholarly journals To Serum or Not to Serum: Reduced-serum Based Approaches for Contact-based Co-culture of Fibroblasts and Keratinocytes for Wound Bed Studies

2020 ◽  
Author(s):  
Snehal Kadam ◽  
Madhusoodhanan Vandana ◽  
Karishma Kaushik
Keyword(s):  
Wound Closure ◽  
Culture Media ◽  
Cell Types ◽  
High Serum ◽  
Dermal Fibroblasts ◽  
Epidermal Keratinocytes ◽  
Serum Concentrations ◽  
Human Epidermal Keratinocytes ◽  
Cell Culture Media ◽  
Structure And Functions

Contact-based co-culture of fibroblasts and keratinocytes is important to study the structure and functions of the wound bed. Co-culture of these two cell types in direct contact with each other has been challenging, requiring high serum concentrations (up to 10%), feeder systems and a range of supplemental factors. These approaches are not only technically demanding, but also present scientific, cost and ethical limitations associated with high-serum concentrations. We have developed two reduced-serum approaches (1-2%) to support contact-based co-culture of human dermal fibroblasts (HDFa) and human epidermal keratinocytes (HaCaT). The two approaches include (1) Specialized cell culture media for each cell type mixed in a 1:1 ratio (KGM+FGM), and (2) Minimal media supplemented with cell-specific growth factors (MEM+GF). Co-culture could be successfully achieved by co-seeding (two cell types were introduced simultaneously), or in a layered fashion (keratinocytes seeded on top of confluent fibroblasts). With wound scratch assays, the co-cultured platforms could demonstrate cell proliferation, migration and wound closure. The reduced-serum conditions developed are simple, easy to formulate and adopt, and based on commonly-available media components. These contact-based co-culture approaches can be leveraged for wound and skin studies, and tissue bioengineering applications, potentially reducing concerns with high-serum formulations.

scholarly journals To Serum or Not to Serum: Reduced-serum based methods for contact-based co-culture of human dermal fibroblasts (HDFa) and epidermal keratinocytes (HaCaT) for wound bed studies

2020 ◽  
Author(s):  
Snehal Kadam ◽  
Madhusoodhanan Vandana ◽  
Karishma Kaushik
Keyword(s):  
Direct Contact ◽  
Cell Types ◽  
High Serum ◽  
Dermal Fibroblasts ◽  
Epidermal Keratinocytes ◽  
Human Dermal Fibroblasts ◽  
Serum Concentrations ◽  
High Concentrations ◽  
Serum Free Conditions ◽  
Structure And Functions

Abstract Background: Contact-based co-culture of human dermal fibroblasts and epidermal keratinocytes is important to study wound bed structure and functions. Co-culture of these two cell types in direct contact with each other has been a long-standing and challenging issue, owing to specific and different serum and growth factor requirements of the two cell types. Existing protocols employ high-serum concentrations (up to 10% FBS), complex cell feeder systems, and a range of supplemental factors. These approaches are not only technically demanding, labor- and material-intensive but also pose scientific and ethical limitations associated with high concentrations of animal serum. On the other hand, serum-free conditions often fail to support the attachment and proliferation of one or both cell types when cultured together. Results: Given this, we have developed two reduced-serum based approaches (1-2% serum), using commonly-available media components, to support the contact-based co-culture of HDFa and HaCaT cells. Using these approaches, HDFa and HaCaT were co-cultured by layering keratinocytes over confluent fibroblasts or by co-seeding the two cell types simultaneously. Under these conditions, both cell types showed robust attachment and proliferation, and characteristic cellular morphology in co-culture. Further, these co-cultured platforms enabled the study of important wound bed functions such as cell migration and wound closure.Conclusions: We believe that these methods can be leveraged for a range of wound and skin studies, and tissue bioengineering applications, potentially reducing concerns with high-serum formulations.

scholarly journals Reduced serum methods for contact-based coculture of human dermal fibroblasts and epidermal keratinocytes

BioTechniques ◽  
2020 ◽  
Vol 69 (5) ◽  
pp. 347-355
Author(s):  
Snehal Kadam ◽  
Madhusoodhanan Vandana ◽  
Karishma S Kaushik
Keyword(s):  
Wound Closure ◽  
Fetal Bovine Serum ◽  
Cell Types ◽  
High Serum ◽  
Dermal Fibroblasts ◽  
Epidermal Keratinocytes ◽  
Human Dermal Fibroblasts ◽  
Fetal Bovine ◽  
High Concentrations ◽  
Serum Free Conditions

Direct contact-based coculture of human dermal fibroblasts and epidermal keratinocytes has been a long-standing and challenging issue owing to different serum and growth factor requirements of the two cell types. Existing protocols employ high serum concentrations (up to 10% fetal bovine serum), complex feeder systems and a range of supplemental factors. These approaches are technically demanding and labor intensive, and pose scientific and ethical limitations associated with the high concentrations of animal serum. On the other hand, serum-free conditions often fail to support the proliferation of one or both cell types when they are cultured together. We have developed two reduced serum approaches (1–2% serum) that support the contact-based coculture of human dermal fibroblasts and immortalized keratinocytes and enable the study of cell migration and wound closure.

Demonstration of a Choline Requirement for Optimal Keratinocyte Growth in a Defined Culture Medium

1988 ◽  
Vol 118 (12) ◽  
pp. 1487-1494 ◽  
Author(s):  
Philip R. Gordon ◽  
Liebe K. Gelman ◽  
Barbara A. Gilchrest
Keyword(s):  
Culture Medium ◽  
Basal Medium ◽  
Donor Cell ◽  
Cell Types ◽  
Dermal Fibroblasts ◽  
Cell Yield ◽  
Epidermal Keratinocytes ◽  
Good Growth ◽  
Human Epidermal Keratinocytes ◽  
Defined Culture

Abstract Nutrient requirements for proliferation and differentiated function of individual cell types can be determined using cell culture methodologies. Human epidermal keratinocytes are stimulated to grow by choline supplementation in the presence of myo-inositol when grown in a commercial nutrient medium containing six other defined supplements. The optimal range of choline concentrations varied among donor cell lines, but consistently fell between 36 µM and 180 µM. Addition of 72 µM choline increased cell yield to 250 ± 38% of that produced by myo-inositol supplementation alone and 92 ± 8% of that produced by addition of a highly mitogenic hypothalamic extract, which was previously required for good growth in this culture system. Supplementation of the basal medium with both the extract and choline resulted in 165 ± 13% of the cell yield observed with the extract addition alone. Supplementation with other phospholipid precursors did not further increase keratinocyte growth. Neither dermal fibroblasts nor epidermal melanocytes were stimulated by supplementation with choline, suggesting the high keratinocyte requirement is unusual. This completely defined culture medium for keratinocyte growth should prove useful in analyzing the role of phospholipids and other nutrients in human epidermis.

Keratinocyte growth regulation in fibroblast cocultures via a double paracrine mechanism

1999 ◽  
Vol 112 (12) ◽  
pp. 1843-1853 ◽  
Author(s):  
N. Maas-Szabowski ◽  
A. Shimotoyodome ◽  
N.E. Fusenig
Keyword(s):  
Neutralizing Antibodies ◽  
Growth Regulation ◽  
Keratinocyte Growth Factor ◽  
Dermal Fibroblasts ◽  
Tissue Homeostasis ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Keratinocyte Proliferation ◽  
Normal Human ◽  
Kinetics Of

Epithelial-mesenchymal interactions play an important role in regulating tissue homeostasis and repair. For skin, the regulatory mechanisms of epidermal-dermal interactions were studied in cocultures of normal human epidermal keratinocytes (NEK) and dermal fibroblasts (HDF) rendered postmitotic by alpha-irradiation (HDFi). The expression kinetics of different cytokines and their receptors with presumed signalling function in skin were determined at the RNA and protein level in mono- and cocultured NEK and HDFi. In cocultured HDFi, mRNA and protein synthesis of keratinocyte growth factor (KGF) (FGF-7) was strongly enhanced, whereas in cocultured keratinocytes interleukin (IL)-1alpha and -1beta mRNA expression increased compared to monocultures. Thus we postulated that IL-1, which had no effect on keratinocyte proliferation, induced in fibroblasts the expression of factors stimulating keratinocyte proliferation, such as KGF. The functional significance of this reciprocal modulation was substantiated by blocking experiments. Both IL-1alpha and -1beta-neutralizing antibodies and IL-1 receptor antagonist significantly reduced keratinocyte proliferation supposedly through abrogation of KGF production, because IL-1 antibodies blocked the induced KGF production. These data indicate a regulation of keratinocyte growth by a double paracrine mechanism through release of IL-1 which induces KGF in cocultured fibroblasts. Thus IL-1, in addition to its proinflammatory function in skin, may play an essential role in regulating tissue homeostasis.

scholarly journals Persea americanaMill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Maria del R. Ramos-Jerz ◽  
Socorro Villanueva ◽  
Gerold Jerz ◽  
Peter Winterhalter ◽  
Alexandra M. Deters
Keyword(s):  
Chlorogenic Acid ◽  
Dermal Fibroblasts ◽  
Persea Americana ◽  
Epidermal Keratinocytes ◽  
Hacat Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Residual Solvent ◽  
Keratinocyte Proliferation ◽  
Normal Human

Methanolic avocado (Persea americanaMill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Theirin vitroinfluence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fractionM.2composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore,M.2increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fractionM.6increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fractionM.7contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes.

Ras‐induced spreading and wound closure in human epidermal keratinocytes

2005 ◽  
Vol 19 (13) ◽  
pp. 1836-1838 ◽  
Author(s):  
Michael Tscharntke ◽  
Ruth Pofahl ◽  
Thomas Krieg ◽  
Ingo Haase
Keyword(s):  
Wound Closure ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes

Effects of Sulfur Mustard on Cytokines Released from Cultured Human Epidermal Keratinocytes

1998 ◽  
Vol 17 (3) ◽  
pp. 223-229 ◽  
Author(s):  
Elien M. Kurt ◽  
Robert J. Schafer ◽  
Carmen M. Arroyo
Keyword(s):  
Sulfur Mustard ◽  
Cell Types ◽  
Epidermal Keratinocytes ◽  
Cytokine Release ◽  
Experimental Conditions ◽  
Human Epidermal Keratinocytes ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Cytokine Levels ◽  
Factor Alpha

The release of the cytokines interleukin (IL)-1α, IL-1β, IL-6, IL-8, and tumor necrosis factor alpha (TNF-α was measured from epiderm alkeratinocytes in an attempt to characterize the immunologic response in keratinocytes following exposure to bis (2-chloroethyl)sulfide (sulfur mustard, HD). Enzyme-linked immunosorbentassay (ELISA) was used to measure cytokine levels in adult and neonatal culture human epidermal keratinocytes (HEK) 3 h after exposure to 0.50 and 1.0 m M HD. A two-way analysis of variance was carried out for cell type and HD concentration. That analysis showed significant differences between cell types for IL-1α and IL-1β(p =.001 and p =.015, respectively). In both of these cytokines, release in neonatal HEK decreased less than in adult HEK. A significant effectof HD concentration was shown only for IL-1β (P <.001), with cytokine release decreasing with increasing HD dose. In addition, a significant cell donor type by HD concentration interaction effect was found for IL-1β under the experimental conditions described in materials and methods. With increasing HD concentration, the relative decrease in cytokine release was much greater for adult than for neonatal HEK.

scholarly journals Ultraviolet B, melanin and mitochondrial DNA: Photo-damage in human epidermal keratinocytes and melanocytes modulated by alpha-melanocyte-stimulating hormone

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 881 ◽  
Author(s):  
Markus Böhm ◽  
Helene Z. Hill
Keyword(s):  
Mitochondrial Dna ◽  
Copy Number ◽  
Cell Types ◽  
Ultraviolet B ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Mtdna Damage ◽  
Melanocyte Stimulating Hormone ◽  
Copy Numbers ◽  
Stimulating Hormone

Alpha-melanocyte-stimulating hormone (alpha-MSH) increases melanogenesis and protects from UV-induced DNA damage. However, its effect on mitochondrial DNA (mtDNA) damage is unknown. We have addressed this issue in a pilot study using human epidermal keratinocytes and melanocytes incubated with alpha-MSH and irradiated with UVB. Real-time touchdown PCR was used to quantify total and deleted mtDNA. The deletion detected encompassed the common deletion but was more sensitive to detection. There were 4.4 times more mtDNA copies in keratinocytes than in melanocytes. Irradiation alone did not affect copy numbers. Alpha-MSH slightly increased copy numbers in both cell types in the absence of UVB and caused a similar small decrease in copy number with dose in both cell types. Deleted copies were nearly twice as frequent in keratinocytes as in melanocytes. Alpha-MSH reduced the frequency of deleted copies by half in keratinocytes but not in melanocytes. UVB dose dependently led to an increase in the deleted copy number in alpha-MSH-treated melanocytes. UVB irradiation had little effect on deleted copy number in alpha-MSH-treated keratinocytes. In summary, alpha-MSH enhances mtDNA damage in melanocytes presumably by increased melanogenesis, while α-MSH is protective in keratinocytes, the more so in the absence of irradiation.

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