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Article ; Online: Tie-Over Bolster Pressure Dressing Improves Outcomes of Skin Substitutes Xenografts on Athymic Mice

Andréanne Cartier / Martin A. Barbier / Danielle Larouche / Amélie Morissette / Ariane Bussières / Livia Montalin / Chanel Beaudoin Cloutier / Lucie Germain

International Journal of Molecular Sciences, Vol 23, Iss 5507, p

2022 Volume 5507

Abstract: The efficacy of skin substitutes is established for the treatment of burn injuries, but its use is not limited to this condition. This technology has the potential to improve the treatment of various conditions by offering highly advanced and ... ...

Abstract	The efficacy of skin substitutes is established for the treatment of burn injuries, but its use is not limited to this condition. This technology has the potential to improve the treatment of various conditions by offering highly advanced and personalized treatments. In vivo studies are challenging but essential to move to clinical use in humans. Mice are the most widely used species in preclinical studies, but the main drawback of this model is the limited surface area of the graft in long-term transplantation studies caused by the displacement and the contraction of the graft. We improved the conventional surgical procedures by stabilizing the chamber covering the graft with intramuscular sutures and by adding a tie-over bolster dressing. The current study was therefore performed to compare outcomes of skin grafts between the conventional and optimized skin graft model. Human self-assembled skin substitutes (SASSs) were prepared and grafted to athymic mice either by the conventional method or by the new grafting method. Graft healing and complications were assessed using digital photographs on postoperative days 7, 14, and 21. Similar structure and organization were observed by histological staining. The new grafting method reduced medium and large displacement events by 1.26-fold and medium and large contraction events by 1.8-fold, leading to a 1.6-fold increase in graft surface area compared to skin substitutes grafted with the usual method. This innovation ensures better reproducibility and consistency of skin substitute transplants on mice.
Keywords	tissue engineering ; skin substitutes ; skin graft ; murine model ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Language	English
Publishing date	2022-05-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: A Newly Developed Chemically Defined Serum-Free Medium Suitable for Human Primary Keratinocyte Culture and Tissue Engineering Applications

Sergio Cortez Ghio / Martin A. Barbier / Emilie J. Doucet / Imad Debbah / Meryem Safoine / Gaëtan Le-Bel / Andréanne Cartier / Emilie Jolibois / Amélie Morissette / Danielle Larouche / Julie Fradette / Sylvain L. Guérin / Alain Garnier / Lucie Germain

International Journal of Molecular Sciences, Vol 24, Iss 1821, p

2023 Volume 1821

Abstract: In our experience, keratinocytes cultured in feeder-free conditions and in commercially available defined and serum-free media cannot be as efficiently massively expanded as their counterparts grown in conventional bovine serum-containing medium, nor can ...

Abstract	In our experience, keratinocytes cultured in feeder-free conditions and in commercially available defined and serum-free media cannot be as efficiently massively expanded as their counterparts grown in conventional bovine serum-containing medium, nor can they properly form a stratified epidermis in a skin substitute model. We thus tested a new chemically defined serum-free medium, which we developed for massive human primary keratinocyte expansion and skin substitute production. Our medium, named Surge Serum-Free Medium (Surge SFM), was developed to be used alongside a feeder layer. It supports the growth of keratinocytes freshly isolated from a skin biopsy and cryopreserved primary keratinocytes in cultured monolayers over multiple passages. We also show that keratin-19-positive epithelial stem cells are retained through serial passaging in Surge SFM cultures. Transcriptomic analyses suggest that gene expression is similar between keratinocytes cultured with either Surge SFM or the conventional serum-containing medium. Additionally, Surge SFM can be used to produce bilayered self-assembled skin substitutes histologically similar to those produced using serum-containing medium. Furthermore, these substitutes were grafted onto athymic mice and persisted for up to six months. In conclusion, our new chemically defined serum-free keratinocyte culture medium shows great promise for basic research and clinical applications.
Keywords	cell culture ; tissue engineering ; defined medium ; stem cells ; skin ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	571
Language	English
Publishing date	2023-01-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Cultured Autologous Corneal Epithelia for the Treatment of Unilateral Limbal Stem Cell Deficiency

Louis-Philippe Guérin / Danielle Larouche / Mohib W. Morcos / Anne Faucher / François A. Auger / Bartha M. Knoppers / Ralph Kyrillos / Richard Bazin / Lucie Germain

Biomedicines, Vol 10, Iss 8, p

A Case Series of 15 Patients

2022 Volume 1958

Abstract: Damage to limbal epithelial stem cells can lead to limbal stem cell deficiency (LSCD). Current autologous treatment procedures for unilateral LSCD bear a significant risk of inducing LSCD in the donor eye. This complication can be avoided by grafting a ... ...

Abstract	Damage to limbal epithelial stem cells can lead to limbal stem cell deficiency (LSCD). Current autologous treatment procedures for unilateral LSCD bear a significant risk of inducing LSCD in the donor eye. This complication can be avoided by grafting a stem cell containing cultured autologous corneal epithelium (CACE). The primary objective of this study was to demonstrate the safety of CACE grafted on eyes with LSCD. The secondary objective was to assess the efficacy of a CACE graft in restoring a self-renewing corneal surface with adequate anatomic structures, as well as improving the best corrected visual acuity (BCVA). Fifteen patients were grafted with a CACE on a fibrin gel produced from a 3 mm 2 limbal biopsy harvested from the donor eye. Data were collected at baseline and after grafting. Follow-ups from 1 to 5 years were conducted. No major adverse events related to the CACE graft were observed. For every visit, an anatomic score based on corneal opacity as well as central vascularization and a functional score based on BCVA were determined. Safety was demonstrated by the low occurrence of complications. Anatomical (93%) and functional (47%) results are promising for improving vision in LSCD patients. Combined functional success and partial success rates with inclusion of BCVA were 53% [CI95: 27–79%] one year after CACE grafting. At the last follow-up, 87% [CI95: 60–98%] of the patients had attained corneal clarity. The outcomes demonstrate the safety of our technique and are promising regarding the efficacy of CACE in these patients.
Keywords	cultivated limbal epithelial transplant CLET ; cornea ; stem cells ; limbal stem cell deficiency/LSCD ; eye ; Biology (General) ; QH301-705.5
Subject code	610
Language	English
Publishing date	2022-08-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Are the Effects of the Cholera Toxin and Isoproterenol on Human Keratinocytes’ Proliferative Potential Dependent on Whether They Are Co-Cultured with Human or Murine Fibroblast Feeder Layers?

Sergio Cortez Ghio / Laurence Cantin-Warren / Rina Guignard / Danielle Larouche / Lucie Germain

International Journal of Molecular Sciences, Vol 19, Iss 8, p

2018 Volume 2174

Abstract: Human keratinocyte culture has provided the means to treat burns, wounds and skin pathologies. To date, to efficiently culture keratinocytes, cells are cultured on an irradiated feeder layer (iFL), either comprising human (iHFL) or murine (i3T3FL) ... ...

Abstract	Human keratinocyte culture has provided the means to treat burns, wounds and skin pathologies. To date, to efficiently culture keratinocytes, cells are cultured on an irradiated feeder layer (iFL), either comprising human (iHFL) or murine (i3T3FL) fibroblasts, and the culture medium is supplemented with a cyclic adenosine monophosphate (cAMP) accumulation inducing agent such as isoproterenol (ISO) or cholera toxin (CT). Previous studies have characterized how the feeder layer type and the cAMP inducer type influence epithelial cells’ phenotype independently from one another, but it is still unknown if an optimal combination of feeder layer and cAMP inducer types exists. We used sophisticated statistical models to search for a synergetic effect of feeder layer and cAMP inducer types on human keratinocytes’ proliferative potential. Our data suggests that, when culturing human keratinocytes, using iHFL over i3T3FL increases population doublings and colony-forming efficiency through signaling pathways involving Ak mouse strain thymoma (Akt, also known as protein kinase B) isoforms 1 to 3, signal transducer and activator of transcription 5 (STAT5), p53, and adenosine monophosphate activated protein kinase α1 (AMPKα1). Both tested cAMP inducers ISO and CT yielded comparable outcomes. However, no significant synergy between feeder layer and cAMP inducer types was detected. We conclude that, to promote human keratinocyte growth in the early passages of culture, co-culturing them with a human feeder layer is preferable to a murine feeder layer.
Keywords	keratinocyte ; feeder layer ; cholera toxin ; isoproterenol ; cell culture ; statistical model ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	571
Language	English
Publishing date	2018-07-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Expression of C4.4A in an In Vitro Human Tissue-Engineered Skin Model

Benedikte Jacobsen / Danielle Larouche / Olivier Rochette-Drouin / Michael Ploug / Lucie Germain

BioMed Research International, Vol

2017 Volume 2017

Abstract: A multi-LU-domain-containing protein denoted C4.4A exhibits a tightly regulated membrane-associated expression in the suprabasal layers of stratified squamous epithelia such as skin and the esophagus, and the expression of C4.4A is dysregulated in ... ...

Abstract	A multi-LU-domain-containing protein denoted C4.4A exhibits a tightly regulated membrane-associated expression in the suprabasal layers of stratified squamous epithelia such as skin and the esophagus, and the expression of C4.4A is dysregulated in various pathological conditions. However, the biological function of C4.4A remains unknown. To enable further studies, we evaluated the expression of C4.4A in monolayer cultures of normal human keratinocytes and in tissue-engineered skin substitutes (TESs) produced by the self-assembly approach, which allow the formation of a fully differentiated epidermis tissue. Results showed that, in monolayer, C4.4A was highly expressed in the centre of keratinocyte colonies at cell-cell contacts areas, while some cells located at the periphery presented little C4.4A expression. In TES, emergence of C4.4A expression coincided with the formation of the stratum spinosum. After the creation of a wound within the TES, C4.4A expression was observed in the suprabasal keratinocytes of the migrating epithelium, with the exception of the foremost leading keratinocytes, which were negative for C4.4A. Our results are consistent with previous data in mouse embryogenesis and wound healing. Based on these findings, we conclude that this human TES model provides an excellent surrogate for studies of C4.4A and Haldisin expressions in human stratified epithelia.
Keywords	Medicine ; R
Subject code	616
Language	English
Publishing date	2017-01-01T00:00:00Z
Publisher	Hindawi Limited
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: 27.12 MHz Radiofrequency Ablation for Benign Cutaneous Lesions

Dong Hyun Kim / Dong Ju Hyun / Raymonde Piquette / Clément Beaumont / Lucie Germain / Danielle Larouche

BioMed Research International, Vol

2016 Volume 2016

Abstract: As surgical and/or ablative modalities, radiofrequency (RF) has been known to produce good clinical outcomes in dermatology. Recently, 27.12 MHz RF has been introduced and has several advantages over conventional 4 or 6 MHz in terms of the precise ... ...

Abstract	As surgical and/or ablative modalities, radiofrequency (RF) has been known to produce good clinical outcomes in dermatology. Recently, 27.12 MHz RF has been introduced and has several advantages over conventional 4 or 6 MHz in terms of the precise ablation and lesser pain perception. We aimed to evaluate the clinical efficacy and safety of 27.12 MHz RF for the treatment of benign cutaneous lesions. Twenty female patient subjects were enrolled. Digital photography and a USB microscope camera were used to monitor the clinical results before one session of treatment with 27.12 MHz RF and after 1 and 3 weeks. Treated lesions included telangiectasias, cherry and spider angiomas, skin tags, seborrheic keratoses, lentigo, milium, dilated pore, acne, piercing hole, and one case of neurofibroma. For vascular lesions, clinical results were excellent for 33.3%, good for 44.4%, moderate for 11.1%, and poor for 11.1%. For nonvascular lesions (epidermal lesions and other benign cutaneous lesions), clinical results were excellent for 48.3%, good for 45.2%, moderate for 3.2%, and poor for 3.2%. No serious adverse events were observed. Mild adverse events reported were slight erythema, scale, and crust. The 27.12 MHz RF treatment of benign vascular and nonvascular lesions appears safe and effective after 3 weeks of follow-up.
Keywords	Medicine ; R
Subject code	616
Language	English
Publishing date	2016-01-01T00:00:00Z
Publisher	Hindawi Limited
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Specialized Living Wound Dressing Based on the Self-Assembly Approach of Tissue Engineering

Laurence Cantin-Warren / Rina Guignard / Sergio Cortez Ghio / Danielle Larouche / François A. Auger / Lucie Germain

Journal of Functional Biomaterials, Vol 9, Iss 3, p

2018 Volume 53

Abstract: There is a high incidence of failure and recurrence for chronic skin wounds following conventional therapies. To promote healing, the use of skin substitutes containing living cells as wound dressings has been proposed. The aim of this study was to ... ...

Abstract	There is a high incidence of failure and recurrence for chronic skin wounds following conventional therapies. To promote healing, the use of skin substitutes containing living cells as wound dressings has been proposed. The aim of this study was to produce a scaffold-free cell-based bilayered tissue-engineered skin substitute (TES) containing living fibroblasts and keratinocytes suitable for use as wound dressing, while considering production time, handling effort during the manufacturing process, and stability of the final product. The self-assembly method, which relies on the ability of mesenchymal cells to secrete and organize connective tissue sheet sustaining keratinocyte growth, was used to produce TESs. Three fibroblast-seeding densities were tested to produce tissue sheets. At day 17, keratinocytes were added onto 1 or 3 (reference method) stacked tissue sheets. Four days later, TESs were subjected either to 4, 10, or 17 days of culture at the air–liquid interface (A/L). All resulting TESs were comparable in terms of their histological aspect, protein expression profile and contractile behavior in vitro. However, signs of extracellular matrix (ECM) digestion that progressed over culture time were noted in TESs produced with only one fibroblast-derived tissue sheet. With lower fibroblast density, the ECM of TESs was almost completely digested after 10 days A/L and lost histological integrity after grafting in athymic mice. Increasing the fibroblast seeding density 5 to 10 times solved this problem. We conclude that the proposed method allows for a 25-day production of a living TES, which retains its histological characteristics in vitro for at least two weeks.
Keywords	culture techniques ; regenerative medicine ; skin equivalent ; tissue culture ; bilayered skin substitutes ; tissue engineering ; skin ulcer ; Biotechnology ; TP248.13-248.65 ; Medicine (General) ; R5-920
Subject code	571
Language	English
Publishing date	2018-09-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Improved Methods to Produce Tissue-Engineered Skin Substitutes Suitable for the Permanent Closure of Full-Thickness Skin Injuries

Danielle Larouche / Laurence Cantin-Warren / Maxime Desgagné / Rina Guignard / Israël Martel / Akram Ayoub / Amélie Lavoie / Robert Gauvin / François A. Auger / Véronique J. Moulin / Lucie Germain

BioResearch Open Access, Vol 5, Iss 1, Pp 320-

2016 Volume 329

Abstract: There is a clinical need for skin substitutes to replace full-thickness skin loss. Our group has developed a bilayered skin substitute produced from the patient's own fibroblasts and keratinocytes referred to as Self-Assembled Skin Substitute (SASS). ... ...

Abstract	There is a clinical need for skin substitutes to replace full-thickness skin loss. Our group has developed a bilayered skin substitute produced from the patient's own fibroblasts and keratinocytes referred to as Self-Assembled Skin Substitute (SASS). After cell isolation and expansion, the current time required to produce SASS is 45 days. We aimed to optimize the manufacturing process to standardize the production of SASS and to reduce production time. The new approach consisted in seeding keratinocytes on a fibroblast-derived tissue sheet before its detachment from the culture plate. Four days following keratinocyte seeding, the resulting tissue was stacked on two fibroblast-derived tissue sheets and cultured at the air–liquid interface for 10 days. The resulting total production time was 31 days. An alternative method adapted to more contractile fibroblasts was also developed. It consisted in adding a peripheral frame before seeding fibroblasts in the culture plate. SASSs produced by both new methods shared similar histology, contractile behavior in vitro and in vivo evolution after grafting onto mice when compared with SASSs produced by the 45-day standard method. In conclusion, the new approach for the production of high-quality human skin substitutes should allow an earlier autologous grafting for the treatment of severely burned patients.
Keywords	autologous ; regenerative medicine ; skin equivalent ; tissue culture ; tissue therapy ; transplants ; Medicine ; R ; Biology (General) ; QH301-705.5
Subject code	571
Language	English
Publishing date	2016-11-01T00:00:00Z
Publisher	Mary Ann Liebert
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Irradiated Human Dermal Fibroblasts Are as Efficient as Mouse Fibroblasts as a Feeder Layer to Improve Human Epidermal Cell Culture Lifespan

Lucie Germain / Sylvain L. Guérin / François A. Auger / Odile Damour / Carolyne Simard-Bisson / Éloise Rochefort / Karine Zaniolo / Danielle Larouche / Amélie Lavoie / Francis Bisson

International Journal of Molecular Sciences, Vol 14, Iss 3, Pp 4684-

2013 Volume 4704

Abstract: A fibroblast feeder layer is currently the best option for large scale expansion of autologous skin keratinocytes that are to be used for the treatment of severely burned patients. In a clinical context, using a human rather than a mouse feeder layer is ... ...

Abstract	A fibroblast feeder layer is currently the best option for large scale expansion of autologous skin keratinocytes that are to be used for the treatment of severely burned patients. In a clinical context, using a human rather than a mouse feeder layer is desirable to reduce the risk of introducing animal antigens and unknown viruses. This study was designed to evaluate if irradiated human fibroblasts can be used in keratinocyte cultures without affecting their morphological and physiological properties. Keratinocytes were grown either with or without a feeder layer in serum-containing medium. Our results showed that keratinocytes grown either on an irradiated human feeder layer or irradiated 3T3 cells (i3T3) can be cultured for a comparable number of passages. The average epithelial cell size and morphology were also similar. On the other hand, keratinocytes grown without a feeder layer showed heavily bloated cells at early passages and stop proliferating after only a few passages. On the molecular aspect, the expression level of the transcription factor Sp1, a useful marker of keratinocytes lifespan, was maintained and stabilized for a high number of passages in keratinocytes grown with feeder layers whereas Sp1 expression dropped quickly without a feeder layer. Furthermore, gene profiling on microarrays identified potential target genes whose expression is differentially regulated in the absence or presence of an i3T3 feeder layer and which may contribute at preserving the growth characteristics of these cells. Irradiated human dermal fibroblasts therefore provide a good human feeder layer for an effective expansion of keratinocytes in vitro that are to be used for clinical purposes.
Keywords	feeder layer ; i3T3 ; Sp1 ; human ; skin ; keratinocyte ; differentiation ; proliferation ; microarray ; gene profiling ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	500
Language	English
Publishing date	2013-02-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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