A Novel Method For Isolation Of Stromal Vascular Fraction From Adipose Tissue

Background: Stromal vascular fraction(SVF)is an important heterogeneous cell population within adipose tissue,which plays an important role in the field of tissue regeneration,such as fat grafting,wound healing etc.At present,the isolation of SVF are mainly divided into two main categories,enzymatic digestion and mechanical methods.Enzymatic digestion is a common method to isolate SVF,while they are timeconsuming,costly and may cause biosafety issues.Mechanical isolation of SVF had low cell yield low cell yield and may affect cell surface markers of SVF,reduce progenitor content in SVF cell populations,and impair the biological function of the cells.There are many problems with existing isolation techniques,so there is currently no standardized method for isolation of SVF.Objective: In this paper,ultrasonic mechanical force was introduced to destroy the tight junction between cells and tissues,homogenize the adipose tissue and increase the total surface area of adipose tissue by using the cavitation effect mediated by ultrasonic force,thus reducing the amount of collagenase during digestion and the separation time.Methods: We applied the single variable method to assess the impact of ultrasonic force,ultrasonic time,digestion time and collagenase dosage on cell yield and cell vitality.The cell yield and vitality of the SVF were evaluated by cell counting and trypan blue staining.Cell components of the freshly isolated SVF were analyzed by flow cytometry.The proliferative capacity and differentiation potential of the SVF were identified.The role of SVF in long-term skin expansion was evaluated by a novel animal model.Result:1.Adipose tissue was subjected to sonication at 95 W-20 k Hz for 30 sec followed by 0.15% collagenase for 30 min.Compared to controls(0.20% collagenase digested for 60 min),this treatment resulted in a significant increase in cell number and cell viability,a decrease in the amount of collagenase and a much shorter isolation time.We named ultrasound assisted SVF isolation(USASI).2.Flow cytometry was used to compare the cellular composition of SVF isolated by the two methods,and it was found that the involvement of ultrasound did not affect the proportion of the major cellular components in the SVF.3.The proliferation capacity of the cells was assessed by the cck-8 method and there was no significant difference between the cells isolated by the USASI method and the control.4.Adipogenic,osteogenic and chondrogenic differentiation induction of passaged cells was performed using differentiation induction medium,and there was no significant difference in multilineage differentiation potential between cells isolated by the USASI method and control.5.The luciferase-targeted adipose tissue was transplanted into the dorsal subcutaneous of Lewis rats,the dorsal skin of rats was expanded,the luc positive cells were found in the expanded skin,and Luc/Dlk1,Luc/PDGFRα positive signal were found in the expanded skin.Conclusion: This study found a new method for isolation of SVF,which can shorten SVF isolation time,reduce collagenase dosage,improve cell yield,and provide a new idea for isolation of SVF without affecting SVF activity and its cellular properties.As an important cell population of adipose tissue,SVF provides a scientific rationale to explain adipose tissue promotes skin regeneration.