Clinical Outcomes of Autologous Melanocyte Transplantation Using Enzymatic Separation in Vitiligo

Clinical Outcomes of Autologous Melanocyte Transplantation Using Enzymatic Separation in Vitiligo | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Clinical Outcomes of Autologous Melanocyte Transplantation Using Enzymatic Separation in Vitiligo Leila Dehghani, Rana Moradian Tehrani, Mehrafarin Fesharaki, Fereshteh Alsahebfosoul, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7930900/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Vitiligo is a chronic depigmenting disorder with limited curative treatments. Autologous melanocyte transplantation has emerged as an effective option in stable vitiligo. This study evaluates the clinical outcomes of enzymatically separated autologous melanocyte transplantation in 85 vitiligo patients. Methods A prospective study was conducted on 85 patients (43 males, 42 females) with stable vitiligo. A 3×1 cm donor skin sample was harvested and enzymatically separated within 3 hours using trypsin. The separated melanocyte-keratinocyte suspension was divided into three equal 1×1 cm portions and transplanted to the recipient sites. Patients were followed up for six months. Results The results showed a mean repigmentation rate of 73%, with no recurrence observed during the follow-up period. Overall, 48% of patients achieved excellent repigmentation (> 75%), while 37% demonstrated moderate improvement (50–75%), and 5% showed poor response (< 20%). Importantly, no major complications or adverse events were reported throughout the study, indicating that the procedure was both effective and well-tolerated. Conclusion Autologous melanocyte transplantation via enzymatic separation is a safe and effective approach for treating stable vitiligo, offering reproducible results with minimal downtime and no relapse. Cell & Tissue Engineering Dermatology Vitiligo Autologous Melanocyte Transplantation Enzymatic Separation Skin Pigmentation Figures Figure 1 Highlights Enzymatic autologous melanocyte transplantation achieved a mean repigmentation rate of 73% with no recurrence during six months of follow-up. The procedure is rapid (under 3 hours) , cost-effective , and eliminates the need for cell culture or specialized laboratory facilities. Nearly half of the patients (48%) achieved excellent repigmentation (>75%) , particularly in facial and neck lesions , which showed the best outcomes. The method demonstrated a favorable safety profile , with no major complications or adverse events reported. This technique offers a simple, reproducible, and accessible surgical option for stable vitiligo, making it practical for clinical use in resource-limited settings. Introduction Vitiligo is an acquired pigmentary disorder characterized by the progressive loss of melanocytes, resulting in well-demarcated depigmented macules and patches on the skin. It affects approximately 0.5–2% of the global population and may be associated with significant psychosocial burden, particularly in visible areas such as the face and hands ( 1 , 2 ). Although topical corticosteroids, calcineurin inhibitors and phototherapy remain the mainstay of treatment, they are often inadequate in patients with stable, resistant vitiligo. In such cases, surgical modalities, including autologous melanocyte transplantation, offer a viable alternative ( 3 , 4 ). Autologous non-cultured melanocyte–keratinocyte suspension transplantation (MKST) is a promising technique that allows for regimentation of stable lesions using a small donor site. The enzymatic separation technique enables rapid and efficient isolation of melanocytes without the need for cell culture, thus reducing cost and time (5–7). This study evaluates the clinical outcomes of enzymatic autologous melanocyte transplantation in 85 patients with stable vitiligo, focusing on regimentation rates, safety, and patient satisfaction. Methods This was a single-center prospective clinical study conducted on 85 patients (43 males and 42 females) aged between 18 and 50 years, diagnosed with stable vitiligo (defined as no new lesions or lesion progression in the past 12 months). Patients with stable vitiligo for ≥ 12 months, Lesions on face, trunk, finger and limbs, no prior surgical treatment were included. The patients were admitted to the Jamea lab of Medical Sciences University of Isfahan, Iran and enrolled in the current study according to the inclusion criteria. This study was approved in ethical committee of Medical Sciences University of Isfahan, Iran. A 3×1 cm sample of normal pigmented skin was harvested from the gluteal region. The sample was placed in sterile HBSS solution(ThermoFisher, 14175053) containing 2% antibiotics (amphotericin B and gentamicin (ThermoFisher, R01510)) and rapidly transported to the cleanroom at 2–8°C. The biopsy was incubated in Dispase II(Sigma-Aldrich, 42613-33-2) solution at a concentration of 2.5 U/mL at 37°C for 3 hours. Following incubation, the epidermis was gently detached from the dermis using sterile forceps. The separated epidermis was incubated in 0.25% TrypLE™ solution(Gibco, 10272625) for 50 minutes at 37°C. Subsequently, trypsin activity was neutralized using a solution containing human insulin and HSA (human serum albumin). The dissociated cells were collected by gentle pipetting followed by centrifugation at 1000 rpm for 5 minutes. The collected cells comprised a mixture of melanocytes and keratinocytes. Cell counting and viability assessment were performed using trypan blue staining and a hemocytometer. Cells were suspended in a final volume of 1.5 mL for injection. The resulting cell suspension was then transplanted onto the pre-prepared lesion site. All procedures were performed under sterile conditions using clinical-grade materials and consumables in a Grade B cleanroom under a laminar flow hood at Medical Sciences University of Isfahan. Follow-up Patients were monitored at one and 6 months, post-procedure. Regimentation was assessed by standardized photographs and physician grading: Excellent: >75%, Good: 50–75%, Fair: 20–50%, Poor: <20% Results The study included a total of 85 patients, comprising 43 males and 42 females, with an age range of 18 to 50 years. The mean follow-up duration for the participants was six months. Regimentation Outcomes and Adverse Events Patients’ demographic data are shown in Table 1. No recurrence or pigment loss was observed during the six-month follow-up period. The best responses were noted in facial and neck lesions, whereas acral areas exhibited the least improvement (Table 2). Regarding safety, no cases of infection, scarring, or post-inflammatory hyperpigmentation were reported. Mild erythema and crusting occurred in some patients but resolved spontaneously within two weeks. Table 1. Demographic and clinical data of study subjects Variable Value Total Patients 85 Male 43 Female 42 Age Range 18–50 years Mean Follow-up Duration 6 months Table 2. Repigmentation rates(%) by Body Area. Face/Neck:87, Trunk:73, Limbs:62, Acral:40. In this study, the overall clinical response to autologous melanocyte transplantation was highly encouraging. Nearly half of the patients (48%) achieved an excellent repigmentation outcome with more than 75% restoration of pigmentation in the treated areas. An additional 37% of patients demonstrated a good response (50–75% repigmentation), highlighting the effectiveness of the enzymatic separation technique in the majority of cases. Only 10% of patients showed a fair response (20–50%), and a small minority (5%) experienced a poor response (<20%). These findings indicate that the procedure can deliver high rates of successful repigmentation with durable results, while non-responders remain a limited fraction of the treated population. Discussion This study highlights that enzymatic autologous melanocyte transplantation is a practical, efficient, and dependable approach for restoring skin color in vitiligo. The procedure is rapid cell separation completed in under three hours not only simplifies the process but also reduces patient waiting time. With a mean repigmentation rate of 73% and no recurrence observed over six months, the results demonstrate strong and lasting improvement, consistent with previous reports showing 60–80% success rates using non-cultured melanocyte suspensions. Importantly, this method eliminates the need for expensive cell culture systems, specialized lab infrastructure, or multiple surgeries, making it both cost-effective and clinically accessible. Our findings align closely with earlier studies such as those by Mulekar et al. (2005), who achieved > 75% repigmentation in 65% of patients( 3 ), and Olsson and Juhlin (1998), who reported 74% success( 4 ). What sets our approach apart is its shorter preparation time, the ability to treat multiple recipient areas from a single donor site, and the high proportion of excellent responders (48%), all achieved without cell culture facilities. Additionally, our analysis confirms that lesion location significantly influences treatment outcome. Areas like the face and neck showed the best repigmentation, likely due to their richer blood supply, thinner skin, and higher hair follicle density, factors that support melanocyte survival and migration. In contrast, acral regions (such as the hands and feet) responded less favorably, possibly because of reduced melanocyte mobility, increased friction, and fewer hair follicles, which limit the natural reservoir for pigment cells. Overall, this technique offers a simple, effective, and reproducible option for treating stable vitiligo, with excellent results and minimal procedural demands marking an important step forward in personalized skin repigmentation therapy. The stability of vitiligo proved to be a crucial factor for successful outcomes in this study. All participants had stable disease for at least 12 months, minimizing the risk of immune-mediated destruction of newly transplanted melanocytes. This approach is consistent with the Vitiligo Working Group’s recommendations, which advise performing surgical interventions only in stable cases to ensure long-lasting results( 5 , 6 ). Compared with cultured melanocyte transplantation, the non-cultured enzymatic technique offers several practical advantages. It allows for faster preparation (completed in under three hours), significantly reduces costs by eliminating the need for culture media or incubators, and features a simpler workflow suitable for minor in-clinic operating setups. Additionally, it carries a lower risk of contamination and fewer regulatory requirements, as no ex vivo cell expansion is involved. A major benefit of this method is its high donor-to-recipient expansion ratio—a single 3×1 cm donor area can effectively treat multiple recipient sites, reducing tissue harvesting and minimizing donor site morbidity. The procedure also demonstrated a favorable safety profile, with no major complications such as infection, scarring, or pigmentary irregularities. Mild redness and crusting occurred in a few cases but resolved spontaneously. Importantly, no relapse was observed during six months of follow-up, underscoring the long-term stability and reliability of the technique in properly selected patients. Participants also reported high satisfaction, noting both the natural cosmetic results and the minimal recovery time further supporting this method as a practical and patient-friendly option for vitiligo management. Conclusion Enzymatic separation and autologous melanocyte transplantation are a safe, efficient, and cost-effective option for treating stable vitiligo. With a 73% mean repigmentation rate and no observed recurrence, this method should be considered a first-line surgical treatment for selected patients. Our study demonstrates that autologous transplantation of melanocytes using the enzymatic separation method is a safe, effective, and accessible surgical option for patients with stable vitiligo. The procedure yielded a mean repigmentation rate of 73% with no significant side effects or relapse over a 6-month period. The approach is especially suitable for patients with localized vitiligo and for centers lacking complex tissue culture facilities. By reducing preparation time and expanding the donor-to-recipient ratio, the technique offers excellent clinical utility in real-world settings. Further long-term studies with larger sample sizes and extended follow-up are recommended to validate durability and assess relapse patterns beyond 6 months. Limitations and Future Directions This study has several limitations. First, the absence of a control group, such as patients treated with phototherapy alone, restricts the ability to directly compare outcomes which is our ongoing project. Second, the follow-up duration was relatively short (six months), which limits assessment of long-term stability of repigmentation. Future randomized controlled trials with larger sample sizes and extended follow-up periods more than12 months are recommended to validate these findings and better evaluate long-term efficacy and safety. Declarations All participants provided written informed consent to participate in the study. References Gauthier Y, Surleve-Bazeille JE (1992) Autologous grafting with noncultured melanocytes: a simplified method for treatment of depigmented lesions. J Am Acad Dermatol 26(2):191–194 Mulekar SV (2003) Melanocyte–keratinocyte cell transplantation for stable vitiligo. Int J Dermatol 42(2):132–136 Mulekar SV (2004) Long-term follow-up study of segmental and focal vitiligo treated by autologous, noncultured melanocyte-keratinocyte cell transplantation. Arch Dermatol 140(10):1211–1215 Olsson MJ, Juhlin L (2002) Long-term follow‐up of leucoderma patients treated with transplants of autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br J Dermatol 147(5):893–904 Mulekar SV, Isedeh P (2013) Surgical interventions for vitiligo: an evidence-based review. Br J Dermatol 169(s3):57–66 Bassiouny D, Esmat S (2018) Autologous non-cultured melanocyte–keratinocyte transplantation in the treatment of vitiligo: patient selection and perspectives. Clinical, Cosmetic and Investigational Dermatology. 26:521–540 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":114948,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 1.\u003c/strong\u003e Clinical photographs of a patient with stable vitiligo showing depigmented lesions on the forearm before treatment (B) and excellent repigmentation (\u0026gt;75%) six months after autologous melanocyte transplantation (A). 1. Vitiligo patch on the forehead before treatment (B) and after(A) six months of autologous melanocyte transplantation showing significant repigmentation. 2. Depigmented lesion on the elbow before treatment (B) and partial repigmentation at six-month follow-up(A). 3. Vitiligo lesion on the knee showing poor baseline pigmentation (B) and moderate improvement after treatment (A). 4. Stable vitiligo patch on the chin before treatment (B) and excellent repigmentation observed six months later(A). 5. Vitiligo involving the eyelids (B) with marked improvement in pigmentation following transplantation (B). 6. Acral vitiligo on the hands showing limited repigmentation response six months after treatment (B) compared with baseline (A).\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7930900/v1/aa2171b20cfe92a0bdb53821.jpg"},{"id":94827252,"identity":"9470b07f-b791-4c72-9c29-20b3ff8a82f8","added_by":"auto","created_at":"2025-10-31 06:56:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":635702,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7930900/v1/6bd53f5a-f1f4-4c45-ae12-c80a9c3b9c96.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eClinical Outcomes of Autologous Melanocyte Transplantation Using Enzymatic Separation in Vitiligo\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eEnzymatic autologous melanocyte transplantation achieved a \u003cstrong\u003emean repigmentation rate of 73%\u003c/strong\u003e with \u003cstrong\u003eno recurrence\u003c/strong\u003e during six months of follow-up.\u003c/li\u003e\n \u003cli\u003eThe procedure is \u003cstrong\u003erapid (under 3 hours)\u003c/strong\u003e\u003cstrong\u003e, \u003cstrong\u003ecost-effective\u003c/strong\u003e\u003c/strong\u003e, and eliminates the need for cell culture or specialized laboratory facilities.\u003c/li\u003e\n \u003cli\u003eNearly \u003cstrong\u003ehalf of the patients (48%)\u003c/strong\u003e achieved \u003cstrong\u003eexcellent repigmentation (\u0026gt;75%)\u003c/strong\u003e, particularly in \u003cstrong\u003efacial and neck lesions\u003c/strong\u003e, which showed the best outcomes.\u003c/li\u003e\n \u003cli\u003eThe method demonstrated a \u003cstrong\u003efavorable safety profile\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e with no major complications or adverse events reported.\u003c/li\u003e\n \u003cli\u003eThis technique offers a \u003cstrong\u003esimple, reproducible, and accessible surgical option\u003c/strong\u003e for stable vitiligo, making it practical for clinical use in resource-limited settings.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eVitiligo is an acquired pigmentary disorder characterized by the progressive loss of melanocytes, resulting in well-demarcated depigmented macules and patches on the skin. It affects approximately 0.5\u0026ndash;2% of the global population and may be associated with significant psychosocial burden, particularly in visible areas such as the face and hands (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough topical corticosteroids, calcineurin inhibitors and phototherapy remain the mainstay of treatment, they are often inadequate in patients with stable, resistant vitiligo. In such cases, surgical modalities, including autologous melanocyte transplantation, offer a viable alternative (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAutologous non-cultured melanocyte\u0026ndash;keratinocyte suspension transplantation (MKST) is a promising technique that allows for regimentation of stable lesions using a small donor site. The enzymatic separation technique enables rapid and efficient isolation of melanocytes without the need for cell culture, thus reducing cost and time (5\u0026ndash;7).\u003c/p\u003e\u003cp\u003eThis study evaluates the clinical outcomes of enzymatic autologous melanocyte transplantation in 85 patients with stable vitiligo, focusing on regimentation rates, safety, and patient satisfaction.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThis was a single-center prospective clinical study conducted on 85 patients (43 males and 42 females) aged between 18 and 50 years, diagnosed with stable vitiligo (defined as no new lesions or lesion progression in the past 12 months). Patients with stable vitiligo for \u0026ge;\u0026thinsp;12 months, Lesions on face, trunk, finger and limbs, no prior surgical treatment were included. The patients were admitted to the Jamea lab of Medical Sciences University of Isfahan, Iran and enrolled in the current study according to the inclusion criteria. This study was approved in ethical committee of Medical Sciences University of Isfahan, Iran.\u003c/p\u003e\u003cp\u003eA 3\u0026times;1 cm sample of normal pigmented skin was harvested from the gluteal region. The sample was placed in sterile HBSS solution(ThermoFisher, 14175053) containing 2% antibiotics (amphotericin B and gentamicin (ThermoFisher, R01510)) and rapidly transported to the cleanroom at 2\u0026ndash;8\u0026deg;C. The biopsy was incubated in Dispase II(Sigma-Aldrich, 42613-33-2) solution at a concentration of 2.5 U/mL at 37\u0026deg;C for 3 hours. Following incubation, the epidermis was gently detached from the dermis using sterile forceps. The separated epidermis was incubated in 0.25% TrypLE\u0026trade; solution(Gibco, 10272625) for 50 minutes at 37\u0026deg;C. Subsequently, trypsin activity was neutralized using a solution containing human insulin and HSA (human serum albumin). The dissociated cells were collected by gentle pipetting followed by centrifugation at 1000 rpm for 5 minutes. The collected cells comprised a mixture of melanocytes and keratinocytes. Cell counting and viability assessment were performed using trypan blue staining and a hemocytometer. Cells were suspended in a final volume of 1.5 mL for injection. The resulting cell suspension was then transplanted onto the pre-prepared lesion site. All procedures were performed under sterile conditions using clinical-grade materials and consumables in a Grade B cleanroom under a laminar flow hood at Medical Sciences University of Isfahan.\u003c/p\u003e\n\u003ch3\u003eFollow-up\u003c/h3\u003e\n\u003cp\u003ePatients were monitored at one and 6 months, post-procedure. Regimentation was assessed by standardized photographs and physician grading: Excellent: \u0026gt;75%, Good: 50\u0026ndash;75%, Fair: 20\u0026ndash;50%, Poor: \u0026lt;20%\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe study included a total of 85 patients, comprising 43 males and 42 females, with an age range of 18 to 50 years. The mean follow-up duration for the participants was six months.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRegimentation Outcomes and Adverse Events\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatients\u0026rsquo; demographic data are shown in Table 1. No recurrence or pigment loss was observed during the six-month follow-up period. The best responses were noted in facial and neck lesions, whereas acral areas exhibited the least improvement (Table 2). Regarding safety, no cases of infection, scarring, or post-inflammatory hyperpigmentation were reported. Mild erythema and crusting occurred in some patients but resolved spontaneously within two weeks.\u003c/p\u003e\n\u003cp\u003eTable 1. Demographic and clinical data of study subjects\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003eVariable\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003eValue\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003eTotal Patients\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003e43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003eAge Range\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003e18\u0026ndash;50 years\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003eMean Follow-up Duration\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 288px;\"\u003e\n \u003cp\u003e6 months\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Repigmentation rates(%) by Body Area. Face/Neck:87, Trunk:73, Limbs:62, Acral:40.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cimg 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\"\u003e\u003c/strong\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, the overall clinical response to autologous melanocyte transplantation was highly encouraging. Nearly half of the patients (48%) achieved an \u003cstrong\u003eexcellent repigmentation outcome\u003c/strong\u003e with more than 75% restoration of pigmentation in the treated areas. An additional 37% of patients demonstrated a \u003cstrong\u003egood response\u003c/strong\u003e (50\u0026ndash;75% repigmentation), highlighting the effectiveness of the enzymatic separation technique in the majority of cases. Only 10% of patients showed a \u003cstrong\u003efair response\u003c/strong\u003e (20\u0026ndash;50%), and a small minority (5%) experienced a \u003cstrong\u003epoor response\u003c/strong\u003e (\u0026lt;20%). These findings indicate that the procedure can deliver high rates of successful repigmentation with durable results, while non-responders remain a limited fraction of the treated population.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study highlights that enzymatic autologous melanocyte transplantation is a practical, efficient, and dependable approach for restoring skin color in vitiligo. The procedure is rapid cell separation completed in under three hours not only simplifies the process but also reduces patient waiting time. With a mean repigmentation rate of 73% and no recurrence observed over six months, the results demonstrate strong and lasting improvement, consistent with previous reports showing 60\u0026ndash;80% success rates using non-cultured melanocyte suspensions. Importantly, this method eliminates the need for expensive cell culture systems, specialized lab infrastructure, or multiple surgeries, making it both cost-effective and clinically accessible.\u003c/p\u003e\u003cp\u003eOur findings align closely with earlier studies such as those by Mulekar et al. (2005), who achieved\u0026thinsp;\u0026gt;\u0026thinsp;75% repigmentation in 65% of patients(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), and Olsson and Juhlin (1998), who reported 74% success(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). What sets our approach apart is its shorter preparation time, the ability to treat multiple recipient areas from a single donor site, and the high proportion of excellent responders (48%), all achieved without cell culture facilities.\u003c/p\u003e\u003cp\u003eAdditionally, our analysis confirms that lesion location significantly influences treatment outcome. Areas like the face and neck showed the best repigmentation, likely due to their richer blood supply, thinner skin, and higher hair follicle density, factors that support melanocyte survival and migration. In contrast, acral regions (such as the hands and feet) responded less favorably, possibly because of reduced melanocyte mobility, increased friction, and fewer hair follicles, which limit the natural reservoir for pigment cells.\u003c/p\u003e\u003cp\u003eOverall, this technique offers a simple, effective, and reproducible option for treating stable vitiligo, with excellent results and minimal procedural demands marking an important step forward in personalized skin repigmentation therapy.\u003c/p\u003e\u003cp\u003eThe stability of vitiligo proved to be a crucial factor for successful outcomes in this study. All participants had stable disease for at least 12 months, minimizing the risk of immune-mediated destruction of newly transplanted melanocytes. This approach is consistent with the Vitiligo Working Group\u0026rsquo;s recommendations, which advise performing surgical interventions only in stable cases to ensure long-lasting results(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCompared with cultured melanocyte transplantation, the non-cultured enzymatic technique offers several practical advantages. It allows for faster preparation (completed in under three hours), significantly reduces costs by eliminating the need for culture media or incubators, and features a simpler workflow suitable for minor in-clinic operating setups. Additionally, it carries a lower risk of contamination and fewer regulatory requirements, as no ex vivo cell expansion is involved.\u003c/p\u003e\u003cp\u003eA major benefit of this method is its high donor-to-recipient expansion ratio\u0026mdash;a single 3\u0026times;1 cm donor area can effectively treat multiple recipient sites, reducing tissue harvesting and minimizing donor site morbidity. The procedure also demonstrated a favorable safety profile, with no major complications such as infection, scarring, or pigmentary irregularities. Mild redness and crusting occurred in a few cases but resolved spontaneously.\u003c/p\u003e\u003cp\u003eImportantly, no relapse was observed during six months of follow-up, underscoring the long-term stability and reliability of the technique in properly selected patients. Participants also reported high satisfaction, noting both the natural cosmetic results and the minimal recovery time further supporting this method as a practical and patient-friendly option for vitiligo management.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eEnzymatic separation and autologous melanocyte transplantation are a safe, efficient, and cost-effective option for treating stable vitiligo. With a 73% mean repigmentation rate and no observed recurrence, this method should be considered a first-line surgical treatment for selected patients.\u003c/p\u003e\u003cp\u003eOur study demonstrates that autologous transplantation of melanocytes using the enzymatic separation method is a safe, effective, and accessible surgical option for patients with stable vitiligo. The procedure yielded a mean repigmentation rate of 73% with no significant side effects or relapse over a 6-month period.\u003c/p\u003e\u003cp\u003eThe approach is especially suitable for patients with localized vitiligo and for centers lacking complex tissue culture facilities. By reducing preparation time and expanding the donor-to-recipient ratio, the technique offers excellent clinical utility in real-world settings.\u003c/p\u003e\u003cp\u003eFurther long-term studies with larger sample sizes and extended follow-up are recommended to validate durability and assess relapse patterns beyond 6 months.\u003c/p\u003e\n\u003ch3\u003eLimitations and Future Directions\u003c/h3\u003e\n\u003cp\u003eThis study has several limitations. First, the absence of a control group, such as patients treated with phototherapy alone, restricts the ability to directly compare outcomes which is our ongoing project. Second, the follow-up duration was relatively short (six months), which limits assessment of long-term stability of repigmentation. Future randomized controlled trials with larger sample sizes and extended follow-up periods more than12 months are recommended to validate these findings and better evaluate long-term efficacy and safety.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAll participants provided written informed consent to participate in the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGauthier Y, Surleve-Bazeille JE (1992) Autologous grafting with noncultured melanocytes: a simplified method for treatment of depigmented lesions. J Am Acad Dermatol 26(2):191\u0026ndash;194\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMulekar SV (2003) Melanocyte\u0026ndash;keratinocyte cell transplantation for stable vitiligo. Int J Dermatol 42(2):132\u0026ndash;136\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMulekar SV (2004) Long-term follow-up study of segmental and focal vitiligo treated by autologous, noncultured melanocyte-keratinocyte cell transplantation. Arch Dermatol 140(10):1211\u0026ndash;1215\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOlsson MJ, Juhlin L (2002) Long-term follow‐up of leucoderma patients treated with transplants of autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br J Dermatol 147(5):893\u0026ndash;904\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMulekar SV, Isedeh P (2013) Surgical interventions for vitiligo: an evidence-based review. Br J Dermatol 169(s3):57\u0026ndash;66\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBassiouny D, Esmat S (2018) Autologous non-cultured melanocyte\u0026ndash;keratinocyte transplantation in the treatment of vitiligo: patient selection and perspectives. Clinical, Cosmetic and Investigational Dermatology. 26:521\u0026ndash;540\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Isfahan University of Medical Sciences","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Vitiligo, Autologous Melanocyte Transplantation, Enzymatic Separation, Skin Pigmentation","lastPublishedDoi":"10.21203/rs.3.rs-7930900/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7930900/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eVitiligo is a chronic depigmenting disorder with limited curative treatments. Autologous melanocyte transplantation has emerged as an effective option in stable vitiligo. This study evaluates the clinical outcomes of enzymatically separated autologous melanocyte transplantation in 85 vitiligo patients.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eA prospective study was conducted on 85 patients (43 males, 42 females) with stable vitiligo. A 3\u0026times;1 cm donor skin sample was harvested and enzymatically separated within 3 hours using trypsin. The separated melanocyte-keratinocyte suspension was divided into three equal 1\u0026times;1 cm portions and transplanted to the recipient sites. Patients were followed up for six months.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe results showed a mean repigmentation rate of 73%, with no recurrence observed during the follow-up period. Overall, 48% of patients achieved excellent repigmentation (\u0026gt;\u0026thinsp;75%), while 37% demonstrated moderate improvement (50\u0026ndash;75%), and 5% showed poor response (\u0026lt;\u0026thinsp;20%). Importantly, no major complications or adverse events were reported throughout the study, indicating that the procedure was both effective and well-tolerated.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eAutologous melanocyte transplantation via enzymatic separation is a safe and effective approach for treating stable vitiligo, offering reproducible results with minimal downtime and no relapse.\u003c/p\u003e","manuscriptTitle":"Clinical Outcomes of Autologous Melanocyte Transplantation Using Enzymatic Separation in Vitiligo","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-30 12:11:03","doi":"10.21203/rs.3.rs-7930900/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"50c95748-a20f-4a1a-ab09-e69c47a858b3","owner":[],"postedDate":"October 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56762177,"name":"Cell \u0026 Tissue Engineering"},{"id":56762178,"name":"Dermatology"}],"tags":[],"updatedAt":"2025-10-30T12:11:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-30 12:11:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7930900","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7930900","identity":"rs-7930900","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}