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Platelet lysate can support the development of a 3D-engineered skin for clinical application.

ABSTRACT: Safety concerns associated with foetal bovine serum (FBS) have restricted its translation into clinics. We hypothesised that platelet lysate (PL) can be utilised as a safe alternative to produce serum-free 3D-engineered skin. PL supported a short-term expansion of fibroblasts, with negligible replication-induced senescence and directed epidermal stratification. PL-expanded fibroblasts were phenotypically separated into three subpopulations of CD90⁺FAP⁺, CD90⁺FAP^- and CD90^-FAP⁺, based on CD90 (reticular marker) and FAP (papillary marker) expression profile. PL drove the expansion of the intermediate CD90⁺ FAP⁺ subpopulation in expense of reticular CD90⁺FAP^-, which may be less fibrotic once grafted. The 3D-engineered skin cultured in PL was analysed by immunofluorescence using specific markers. Detection of ColIV and LMN-511 confirmed basement membrane. K10 confirmed near native differentiation pattern of neo-epidermis. CD29- and K5-positive interfollicular stem cells were also sustained. Transmission and scanning electron microscopies detailed the ultrastructure of the neo-dermis and neo-epidermis. To elucidate the underlying mechanism of the effect of PL on skin maturation, growth factor contents in PL were measured, and TGF-β1 was identified as one of the most abundant. TGF-β1 neutralising antibody reduced the number of Ki67-positive proliferative cells, suggesting TGF-β1 plays a role in skin maturation. Moreover, the 3D-engineered skin was exposed to lucifer yellow on days 1, 3 and 5. Penetration of lucifer yellow into the skin was used as a semi-quantitative measure of improved barrier function over time. Our findings support the concept of PL as a safe and effective serum alternative for bioengineering skin for cell therapies.

SUBMITTER: Banakh I

PROVIDER: S-EPMC9839813 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Platelet lysate can support the development of a 3D-engineered skin for clinical application.

Banakh I I Rahman Md M MM Arellano C L CL Marks D C DC Mukherjee S S Gargett C E CE Cleland H H Akbarzadeh S S

Cell and tissue research 20221022 1

Safety concerns associated with foetal bovine serum (FBS) have restricted its translation into clinics. We hypothesised that platelet lysate (PL) can be utilised as a safe alternative to produce serum-free 3D-engineered skin. PL supported a short-term expansion of fibroblasts, with negligible replication-induced senescence and directed epidermal stratification. PL-expanded fibroblasts were phenotypically separated into three subpopulations of CD90+FAP+, CD90+FAP< ...[more]

PMID: 36271300

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Project description:BackgroundA significant number of platelet concentrates (PCs) is discarded daily in blood banks due to limited shelf life. Human platelet lysate (HPL), derived from expired PCs, has gained attention as an ethical and sustainable cell culture media supplement in biomedical research and cell therapy production. However, HPL is subject to decisive disadvantages such as batch differences and lack of storage stability. To overcome these limitations and to enhance the applicability of HPL, we developed an HPL manufacturing protocol including a lyophilization process. The aim of this study was to investigate the influence of HPL lyophilization on parameters of quality control, including growth factor concentrations and the culture of human mesenchymal stromal cells (hMSCs).MethodsWe performed a paired comparison of six batches of HPL and lyophilized HPL (L-HPL) regarding the quality parameters pH, total protein, osmolality, sodium, potassium and chloride concentration. Concentrations of 11 growth factors and cytokines were compared between HPL and L-HPL. Additionally, we determined cell yield, proliferation capacity, viability and trilineage differentiation potential of hMSCs following expansion in HPL- and L-HPL-supplemented cell culture media.ResultsQuantification of the quality parameters revealed non-altered pH, osmolality and potassium concentrations and slightly lower total protein, sodium and chloride concentrations of L-HPL compared to HPL. Growth factor and cytokine concentrations did not differ between HPL and L-HPL. Cell yield, division cycles and viability of hMSCs cultured in either HPL- or L-HPL-containing media were comparable. Cells differentiated in medium containing L-HPL showed a slightly higher capacity for osteogenic differentiation, while adipogenic differentiation and chondrogenic differentiation potentials remained unchanged.ConclusionWe successfully developed a method to produce well-applicable L-HPL. The comparison of L-HPL with HPL did not reveal any relevant differences regarding quality control parameters of routine testing, growth factor concentrations and hMSC functionality, demonstrating the suitability of L-HPL as a cell culture supplement. These results emphasize the potential of L-HPL as a sustainable and ethical alternative to animal-derived serum products in biomedical research and drug development.

Project description:The mechanobiological behavior of mesenchymal stem cells (MSCs) in two- (2D) or three-dimensional (3D) cultures relies on the formation of actin filaments which occur as stress fibers and depends on mitochondrial dynamics involving vimentin intermediate filaments. Here we investigate whether human platelet lysate (HPL), that can potentially replace fetal bovine serum for clinical-scale expansion of functional cells, can modulate the stress fiber formation, alter mitochondrial morphology, change membrane elasticity and modulate immune regulatory molecules IDO and GARP in amnion derived MSCs. We can provide evidence that culture supplementation with HPL led to a reduction of stress fiber formation in 2D cultured MSCs compared to a conventional growth medium (MSCGM). 3D MSC cultures, in contrast, showed decreased actin concentrations independent of HPL supplementation. When stress fibers were further segregated by their binding to focal adhesions, a reduction in ventral stress fibers was observed in response to HPL in 2D cultured MSCs, while the length of the individual ventral stress fibers increased. Dorsal stress fibers or transverse arcs were not affected. Interestingly, ventral stress fiber formation did not correlate with membrane elasticity. 2D cultured MSCs did not show differences in the Young's modulus when propagated in the presence of HPL and further cultivation to passage 3 also had no effect on membrane elasticity. In addition, HPL reduced the mitochondrial mass of 2D cultured MSCs while the mitochondrial mass in 3D cultured MSCs was low initially. When mitochondria were segregated into punctuate, rods and networks, a cultivation-induced increase in punctuate and network mitochondria was observed in 2D cultured MSCs of passage 3. Finally, mRNA and protein expression of the immunomodulatory molecule IDO relied on stimulation of 2D culture MSCs with pro-inflammatory cytokines IFN-? and TNF-? with no effect upon HPL supplementation. GARP mRNA and surface expression was constitutively expressed and did not respond to HPL supplementation or stimulation with IFN-? and TNF-?. In conclusion, we can say that MSCs cultivated in 2D and 3D are sensitive to medium supplementation with HPL with changes in actin filament formation, mitochondrial dynamics and membrane elasticity that can have an impact on the immunomodulatory function of MSCs.

Project description:BackgroundThe autologous nerve graft, despite its donor site morbidity and unpredictable functional recovery, continues to be the gold standard in peripheral nerve repair. Rodent research studies have shown promising results with cell transplantation of human adipose-derived stem cells (hADSC) in a bioengineered conduit, as an alternative strategy for nerve regeneration. To achieve meaningful clinical translation, cell therapy must comply with biosafety. Cell extraction and expansion methods that use animal-derived products, including enzymatic adipose tissue dissociation and the use of fetal bovine serum (FBS) as a culture medium supplement, have the potential for transmission of zoonotic infectious and immunogenicity. Human-platelet-lysate (hPL) serum has been used in recent years in human cell expansion, showing reliability in clinical applications.MethodsWe investigated whether hADSC can be routinely isolated and cultured in a completely xenogeneic-free way (using hPL culture medium supplement and avoiding collagenase digestion) without altering their physiology and stem properties. Outcomes in terms of stem marker expression (CD105, CD90, CD73) and the osteocyte/adipocyte differentiation capacity were compared with classical collagenase digestion and FBS-supplemented hADSC expansion.ResultsWe found no significant differences between the two examined extraction and culture protocols in terms of cluster differentiation (CD) marker expression and stem cell plasticity, while hADSC in hPL showed a significantly higher proliferation rate when compared with the usual FBS-added medium. Considering the important key growth factors (particularly brain-derived growth factor (BDNF)) present in hPL, we investigated a possible neurogenic commitment of hADSC when cultured with hPL. Interestingly, hADSC cultured in hPL showed a statistically higher secretion of neurotrophic factors BDNF, glial cell-derived growth factor (GDNF), and nerve-derived growth factor (NFG) than FBS-cultured cells. When cocultured in the presence of primary neurons, hADSC which had been grown under hPL supplementation, showed significantly enhanced neurotrophic properties.ConclusionsThe hPL-supplement medium could improve cell proliferation and neurotropism while maintaining stable cell properties, showing effectiveness in clinical translation and significant potential in peripheral nerve research.

Project description:BackgroundCurrent treatments for several corneal lesions show limited efficacy. Here we report the clinical evaluation of the efficacy of a novel eye drop preparation produced in a public cord blood (CB) bank.Materials and methodsIn a multicentre, retrospective, consecutive case study we evaluated 33 patients (46 eyes) unresponsive to conventional treatments who required urgent intervention. The patients were given allogeneic eye drops obtained from cord blood platelet lysate (CBED) to treat severe ocular surface lesions under a compassionate use protocol. The CBED were prepared from CB units donated for haematopoietic stem cell transplantation that did not contain the minimum stem cell dose required for this use. Patients were grouped by acute conditions (neurotrophic ulcers: group I; other corneal ulcers: group II; corneal burns: group III), and chronic conditions (ocular graft-versus-host disease: group IV; severe dry eye syndrome: group V). The patients received one or two drops of the product to the affected eye four to six times per day for 19 days. A further 19-day cycle of treatment could be repeated according to the initial clinical response.ResultsPatients received a median of 19 CBED vials (interquartile range 19-57, range 19-442) to complete the therapy. Group I-II-III patients showed full and partial ulcer recovery in 25 (78%) and six (19%) eyes respectively. One eye (3%) did not respond to treatment. For groups IV-V improvement was reported for 12 (85%) eyes and lesions worsened on treatment in both eyes (15%) of one patient. No severe adverse events were directly attributed to CBED.DiscussionPromptly available CBED resulted in a well-tolerated allogeneic treatment that showed evidence of efficacy in this cohort of patients. These positive results support further studies on CBED from platelet lysate as a novel product of CB banks. A prospective clinical trial in neurotrophic keratitis (NCT03084861) is ongoing to confirm these preliminary data.

Project description:BackgroundAmong the emerging treatments for erectile dysfunction (ED), platelet-rich plasma (PRP), known for its ability to enhance tissue repair and regeneration, stands out as a promising therapeutic approach. In this innovative study, we aimed to assess the efficacy of intracavernous injections of platelet lysate (PL), a derivative of PRP, in improving erectile function among ED patients.MethodsWe enrolled twenty-six patients, aged between 35 and 70 years (mean age 51.6 ± 11.3 years), who had been experiencing ED for over six months and had an International Index of Erectile Function-5 (IIEF-5) score of 21 or less. Participants received autologous PL injections intracavernously every two weeks for a total of five administrations. We assessed Erection Hardness Score (EHS) and International Index of Erectile Function-5 (IIEF-5) bi-weekly for 16 weeks and conducted penile Doppler ultrasounds pre- and post-treatment to record peak systolic velocity (PSV) and resistance index (RI).ResultsBefore treatment, the mean EHS was 2.15 ± 0.88 and IIEF-5 was 10.92 ± 5.28. Remarkable improvements were observed post-treatment, with the EHS significantly increasing to 3.15 ± 0.83 (p < 0.05) and IIEF-5 to 17.23 ± 5.26 (p < 0.05). Penile Doppler ultrasound exhibited an increase in both PSV and RI post-treatment, with the rise in RI being statistically significant.ConclusionsOur findings indicate that intracavernous injections of PL substantially enhance erectile function, as evidenced by improvements in EHS, IIEF-5, and the RI of penile Doppler ultrasound, without hemorrhagic events or other adverse reactions apart from temporary pain at the injection site during the 16-week follow-up period. These encouraging results suggest that PL injections are a safe and effective treatment modality for patients with moderate ED, potentially providing a less invasive and more physiologically friendly alternative to current ED management strategies.Trial registrationThe study received approval from the Institutional Review Board of National Taiwan University Hospital (IRB Number 202008061RIPC, date of registration 08/28/2020).

Project description:Pressure ulcers (PU) are caused by persistent long-term pressure, which compromises the integrity of the epidermis, dermis, and subcutaneous adipose tissue layer by layer, making it difficult to heal. Platelet products such as platelet lysate (PL) can promote tissue regeneration by secreting numerous growth factors based on clinical studies on skin wound healing. However, the components of PL are difficult to retain in wounds. Gelatin methacrylate (GelMA) is a photopolymerizable hydrogel that has lately emerged as a promising material for tissue engineering and regenerative medicine. The PL liquid was extracted, flow cytometrically detected for CD41a markers, and evenly dispersed in the GelMA hydrogel to produce a surplus growth factor hydrogel system (PL@GM). The microstructure of the hydrogel system was observed under a scanning electron microscope, and its sustained release efficiency and biological safety were tested in vitro. Cell viability and migration of human dermal fibroblasts, and tube formation assays of human umbilical vein endothelial cells were applied to evaluate the ability of PL to promote wound healing and regeneration in vitro. Real-time polymerase chain reaction (PCR) and western blot analyses were performed to elucidate the skin regeneration mechanism of PL. We verified PL's therapeutic effectiveness and histological analysis on the PU model. PL promoted cell viability, migration, wound healing and angiogenesis in vitro. Real-time PCR and western blot indicated PL suppressed inflammation and promoted collagen I synthesis by activating STAT3. PL@GM hydrogel system demonstrated optimal biocompatibility and favorable effects on essential cells for wound healing. PL@GM also significantly stimulated PU healing, skin regeneration, and the formation of subcutaneous collagen and blood vessels. PL@GM could accelerate PU healing by promoting fibroblasts to migrate and secrete collagen and endothelial cells to vascularize. PL@GM promises to be an effective and convenient treatment modality for PU, like chronic wound treatment.

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Platelet lysate can support the development of a 3D-engineered skin for clinical application.

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Platelet lysate can support the development of a 3D-engineered skin for clinical application.

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