Brief comparative study on the isolation and culture methods of human keratinocyte from skin tissue

Nurhayati, Retno Wahyu; Sandora, Normalina; Suwarti,; Nauli, Raisa; Khoiriyah, Zakiyah; Wardhana, Aditya

doi:10.1063/1.5139332

Cited by 2 publications

(7 citation statements)

References 14 publications

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“…The risk of contamination by fibroblasts is theoretically more important with the direct explant technique 6 . However, the use of an adapted medium, low in calcium, allows to create a negative selectivity and to inhibit their growth 16,25 . We did not note any contamination by fibroblasts in our cultures.…”

Section: Discussionmentioning

confidence: 68%

“…6 However, the use of an adapted medium, low in calcium, allows to create a negative selectivity and to inhibit their growth. 16,25 We did not note any contamination by fibroblasts in our cultures.…”

Section: Discussionmentioning

confidence: 71%

“…Indeed, most of the enzymatic method protocols show favourable results when applied on large size samples from abdominoplasties or foreskins, allowing to obtain samples of 5-6 cm 2 or more. 8,24 The poor dissociation of the epidermis during the second enzymatic digestion by TrypLE, despite mechanical intervention (tissue dissociator, trituration), 16 drastically decreased the KCs available for seeding. Moreover, as already described, 13,17,25 this method applied to 0.5 cm 2 samples, does not allow to harvest enough KC to guarantee enough density for the initiation of culture.…”

Section: Discussionmentioning

confidence: 99%

“…This feeder layer was composed of X‐irradiated mouses 3 T3 fibroblasts, which support keratinocytes in culture. Although this technique offers a rapid proliferation rate and resistance to apoptosis, it is not recommended for clinical use 7,13,16 . This method has been replaced by defined serum‐free, low‐calcium media and non‐animal feeder layer systems, adapted to clinical requirements 7,13 .…”

Section: Introductionmentioning

confidence: 99%

“…Although this technique offers a rapid proliferation rate and resistance to apoptosis, it is not recommended for clinical use. 7,13,16 This method has been replaced by defined serum-free, low-calcium media and non-animal feeder layer systems, adapted to clinical requirements. 7,13 Different culture methods are described: the enzymatic method which aims to isolate keratinocytes from tissues by using enzymatic digestion 14 and the direct explant method, based on the placement of samples directly in the culture dish.…”

Section: Introductionmentioning

confidence: 99%

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Comparative study of keratinocyte primary culture methods from paediatric skin biopsies for RNA‐sequencing

Salik

Kaderi

Hans

et al. 2022

Experimental Dermatology

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Background Keratinocyte culture is a standard method used to study gene expression, cell differentiation and proliferation. Numerous protocols exist, however their application is frequently unsuitable for small specimens, such as 4‐mm punch skin biopsies. Aims This study compared 3 different methods of keratinocyte culture from paediatric skin biopsies to evaluate which one ensures adequate cell growth for RNA extraction and sequencing. Materials and methods Thirty‐six skin samples were obtained from 4‐mm punch skin biopsies from residual human body material from healthy children. They were cultured in vitro according to 3 different methods: enzymatic method, epidermis explant and direct explant method. Keratinocytes were characterized by immunocytochemistry using pan‐cytokeratin. RNA extraction was performed with RNeasy Mini kit. Quantity and quality of the extracted RNA was assessed to meet the requirements of library preparation for sequencing. Results The direct explant method had largely shown its superiority over the two other methods, with a 100% success rate and an average of 15 days of culture. RNA extraction yielded a mean of 8545.85 ng of RNA per sample with an RQN of 10. Cover‐clip immunochemistry staining with pan‐cytokeratin had confirmed the absence of fibroblast contamination. Discussion Although the enzymatic method is the most frequently used for keratinocyte culture, it is not suitable small samples required in dermatology. The direct explant method guarantees a high growth rate and the extraction of high quality RNA. Variation in the amount of RNA harvested are related to inter‐ and intra‐individual variations and to the conditions of the experiment. Conclusion This study allowed to conclude that the direct explant method is the most efficient and easy method to ensure cell growth when the samples are from 4‐mm punch skin biopsies. This technique avoids fibroblasts contamination and obtains a sufficient quantity and quality of RNA to sequence it.

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Section: Discussionmentioning

confidence: 68%

Section: Discussionmentioning

confidence: 71%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Comparative study of keratinocyte primary culture methods from paediatric skin biopsies for RNA‐sequencing

Salik

Kaderi

Hans

et al. 2022

Experimental Dermatology

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The Effect of Umbilical Cord Blood Serum and Platelet-Rich Plasma Coatings on the Characteristics of Poly(?-caprolactone) Scaffolds for Skin Tissue Engineering Applications

Nurhayati,

Laksono,

Salwa

et al. 2023

IJTech

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The need for effective artificial skin as a substitute for damaged skin in chronic wound therapy is recently growing. Poly(ε-caprolactone) (PCL) has been identified as a potential material for artificial skin scaffolds due to its exceptional mechanical properties and biocompatibility. However, PCL lacks sufficient bioactivity, necessitating the introduction of bioactive molecules to scaffolds. Human umbilical cord blood serum (UCBS) and platelet-rich plasma (PRP), rich in bioactive molecules, are promising coating materials for PCL-based scaffolds. Therefore, this research aimed to investigate the effect of UCBS and PRP coatings on the mechanical properties, cytotoxicity, and cell attachment ability of PCL scaffolds. Scaffolds prepared through glutaraldehyde-mediated cross-linking of 20% (w/v) PCL followed by freeze-drying were immersed with UCBS or PRP overnight. Coating scaffolds with UCBS generated a significantly lower Young's modulus (0.20 MPa) compared to non-coated counterparts (0.27 MPa), while PRP-coated scaffolds showed no substantial change (0.24 MPa). Both UCBS and PRP coatings significantly increased (p < 0.05) the viability and attachment of primary human fibroblast cells on scaffolds, showing the potential to enhance PCL cytocompatibility for artificial skin.

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Brief comparative study on the isolation and culture methods of human keratinocyte from skin tissue

Cited by 2 publications

References 14 publications

Comparative study of keratinocyte primary culture methods from paediatric skin biopsies for RNA‐sequencing

Comparative study of keratinocyte primary culture methods from paediatric skin biopsies for RNA‐sequencing

The Effect of Umbilical Cord Blood Serum and Platelet-Rich Plasma Coatings on the Characteristics of Poly(?-caprolactone) Scaffolds for Skin Tissue Engineering Applications

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