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The impact of short-term refrigeration of human lipoaspirate on adipose-derived stem cells and adipocytes
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MODEL
Journal of Plastic, Reconstructive & Aesthetic Surgery (2014) xx, 1e3
CORRESPONDENCE AND COMMUNICATION The impact of short-term refrigeration of human lipoaspirate on adipose-derived stem cells and adipocytes* Dear Sir, Both liposuction and fat grafting are popular procedures in aesthetic surgery. Current clinical practice is that fat grafting has to be conducted immediately after liposuction in the concern that cell death in lipoaspirate could increase as the storage time is prolonged. However, there is no scientific data in the literature either to support or oppose that concern. It has been a strong desire of both surgeons and patients to be able to preserve lipoaspirate for potential future applications. In our previous studies,1e3 the viability of adipose-derived stem cells (ASCs) and adipocytes in both fresh and deep-frozen lipoaspirates has been examined and cell apoptosis and necrosis have been quantified by Annexin-V/PI assay and analyzed by flow cytometer. The purpose for the present study was to determine the impact of short-term refrigeration of lipoaspirate on ASCs and adipocytes. Two quantitated functional assays were selected to determine cell viability based on the concept that viable cell does not necessarily mean functional, but the functional cell must be viable. Human lipoaspirates were harvested using standard technique. Each lipoaspirate was divided into 5 portions with 5 ml of each. The fresh portion was processed immediately and served as control. Other portions were stored in the refrigerator (2e8 C) and processed at 24 h, 48 h, 72 h or 96 h later respectively. The viability of ASCs was determined by the number of ASC after 24 h culture of stromal vascular fraction (SVF). The viability of adipocytes was assessed by glycerol-3-phosphate dehydrogenase (G3PDH) activity which is widely used to evaluate the biosynthesis of fat in adipocytes. The sterility of lipoaspirate was assessed by bacterial culture using LB Agar powder and inoculating loop. * This study was orally presented in the 11th Annual Meeting of International Federation for Adipose Therapeutics and Science (IFATS) November 21e24, 2013, New York, NY.
All of the participants (n Z 16) were adults (14 females and 2 males). Average age of the participants was 52 3.8 years (SEM) and the average of body mass index was 26 1.6. Liposuction sites were located in hip (n Z 13), thigh (n Z 4) and arm (n Z 2). The average number of ASC in fresh samples was 523,000 88,399. Average number of ASC in cold-stored samples was slightly decreased as the storage time increased. There was no statistically significant difference on ASC number between the fresh and coldstored samples (Figure 1). The average of G3PDH activity was 0.22 0.02 in fresh samples, but decreased slightly in the cold-stored samples as the storage time was extended. The significant difference was found in the samples stored in refrigerator for 96 h (P < 0.05) as it compared to the fresh samples (Figure 2). No bacteria were detected in any of 10 cold-stored lipoaspirates. Comparing to long-term deep-freezing of lipoaspirate, the effect of short-term refrigeration of lipoaspirate has not been investigated extensively. Matsumoto et al.4 reported that cold-storage of lipoaspirats for 3 days reduces ASC yield, but not ASC biological properties. Eom et al.5 found that ASC population was not reduced after 36 h storage of lipoaspirates at 4 C. Our results seems more correlate to the findings from Eom et al. However, the experiment methods among these studies were different. Many factors could affect the results including patient age, sex, body mass index, medical conditions, liposuction locations and techniques, etc. However, in the present study, the influence of these factors has been largely diminished or controlled by our experimental design because all 5 portions of lipoaspirates came from the same individual. Therefore, we can make an apple-to-apple comparison. One of the unique characteristics of ASCs is that they can adhere to the plastic surface. The non-adherent cells (either non-ASCs or dead ASCs) in the flask were removed by PBS washing after 24 h of SVF culture. Our previous studies1e3 have shown that most of cells adherent to the plastic surface after 24 h SVF culture were viable (negative for both Annexin V-FITC and PI) and the ethanol-treated dead ASCs were unable to adhere to the flasks. Therefore, adherent cells from SVF culture not only can be defined as ASCs but also as viable ASCs. The controversy for the present study could be the clinical implications for using refrigerated lipoaspirate. This is the issue regarding how to deal with the remaining lipoaspirate after fat
http://dx.doi.org/10.1016/j.bjps.2014.08.070 1748-6815/ª 2014 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wang WZ, et al., The impact of short-term refrigeration of human lipoaspirate on adipose-derived stem cells and adipocytes, Journal of Plastic, Reconstructive & Aesthetic Surgery (2014), http://dx.doi.org/10.1016/j.bjps.2014.08.070
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MODEL
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Figure 1 storage.
Correspondence and communication
The number of adherent adipose-derived stem cell (SEM) in the lipoaspirates after 24 h, 48 h, 72 h and 96 h of cold-
Figure 2 G3PDH activity in the lipoaspirates after 24 h, 48 h, 72 h and 96 h of cold-storage. *Indicates statistically significant differences (P < 0.05) between fresh and cold-stored lipoaspirates.
grafting procedure. The unused lipoaspirate can be discarded or keep it temporarily in a refrigerator just in case you may need it in next a few days. It is rare but not impossible that an undercorrection or asymmetry in the early post graft period may be found when the patient revisits. Having readily available refrigerated lipoaspirates would be desirable for both surgeons and patients for the potential correction or secondary correction at least it is harmless. In summary, we report that human lipoaspirates stored in refrigerator for first 2e3 days after liposuction are still relatively good quality and sterile. This lays the groundwork for potential injection of refrigerated lipoaspirates. However, this result may need to be verified by an in-vivo study using refrigerated lipoaspirates.
Funding None.
Conflict of interest None.
Ethical approval The Institutional Review Board of the University of Nevada, Reno approved all of the protocols involving human tissue and cells.
Please cite this article in press as: Wang WZ, et al., The impact of short-term refrigeration of human lipoaspirate on adipose-derived stem cells and adipocytes, Journal of Plastic, Reconstructive & Aesthetic Surgery (2014), http://dx.doi.org/10.1016/j.bjps.2014.08.070
+
MODEL
Correspondence and communication
References 1. Wang WZ, Fang XH, Williams SJ, et al. Analysis for apoptosis and necrosis on adipocytes, stromal vascular fraction and adipose-derived stem cells in human lipoaspirates after liposuction. Plast Reconstr Surg 2013;131:77ce85c. 2. Wang WZ, Fang XH, Stephenson LL, et al. The effect of lipoaspirates cryopreservation on adipose-derived stem cells. Aesthet Surg J 2013;33(7):1046e55. 3. Wang WZ, Fang XH, Williams SJ, et al. Lidocaine-induced ASC apoptosis (tumescent vs. local anesthesia). Aesthet Plast Surg 2014. http://dx.doi.org/10.1007/s00266-014-0387-2. 4. Matsumoto D, Shigeura T, Sato K, et al. Influences of preservation at various temperatures on liposuction aspirates. Plast Reconstr Surg 2007;120:1510e7. 5. Eom YW, Lee JE, Yang MS, et al. Rapid isolation of adipose tissue-derived stem cells by the storage of lipoaspirates. Yonsei Med J 2011;52(6):999e1007.
3 Wei Z. Wang Xin-Hua Fang Shelley J. Williams Linda L. Stephenson Richard C. Baynosa Nolan Jaeger Kayvan T. Khiabani William A. Zamboni Department of Surgery, Division of Plastic Surgery, University of Nevada School of Medicine, Las Vegas, NV, USA E-mail address: [email protected] 14 March 2014
Please cite this article in press as: Wang WZ, et al., The impact of short-term refrigeration of human lipoaspirate on adipose-derived stem cells and adipocytes, Journal of Plastic, Reconstructive & Aesthetic Surgery (2014), http://dx.doi.org/10.1016/j.bjps.2014.08.070
MODEL
Journal of Plastic, Reconstructive & Aesthetic Surgery (2014) xx, 1e3
CORRESPONDENCE AND COMMUNICATION The impact of short-term refrigeration of human lipoaspirate on adipose-derived stem cells and adipocytes* Dear Sir, Both liposuction and fat grafting are popular procedures in aesthetic surgery. Current clinical practice is that fat grafting has to be conducted immediately after liposuction in the concern that cell death in lipoaspirate could increase as the storage time is prolonged. However, there is no scientific data in the literature either to support or oppose that concern. It has been a strong desire of both surgeons and patients to be able to preserve lipoaspirate for potential future applications. In our previous studies,1e3 the viability of adipose-derived stem cells (ASCs) and adipocytes in both fresh and deep-frozen lipoaspirates has been examined and cell apoptosis and necrosis have been quantified by Annexin-V/PI assay and analyzed by flow cytometer. The purpose for the present study was to determine the impact of short-term refrigeration of lipoaspirate on ASCs and adipocytes. Two quantitated functional assays were selected to determine cell viability based on the concept that viable cell does not necessarily mean functional, but the functional cell must be viable. Human lipoaspirates were harvested using standard technique. Each lipoaspirate was divided into 5 portions with 5 ml of each. The fresh portion was processed immediately and served as control. Other portions were stored in the refrigerator (2e8 C) and processed at 24 h, 48 h, 72 h or 96 h later respectively. The viability of ASCs was determined by the number of ASC after 24 h culture of stromal vascular fraction (SVF). The viability of adipocytes was assessed by glycerol-3-phosphate dehydrogenase (G3PDH) activity which is widely used to evaluate the biosynthesis of fat in adipocytes. The sterility of lipoaspirate was assessed by bacterial culture using LB Agar powder and inoculating loop. * This study was orally presented in the 11th Annual Meeting of International Federation for Adipose Therapeutics and Science (IFATS) November 21e24, 2013, New York, NY.
All of the participants (n Z 16) were adults (14 females and 2 males). Average age of the participants was 52 3.8 years (SEM) and the average of body mass index was 26 1.6. Liposuction sites were located in hip (n Z 13), thigh (n Z 4) and arm (n Z 2). The average number of ASC in fresh samples was 523,000 88,399. Average number of ASC in cold-stored samples was slightly decreased as the storage time increased. There was no statistically significant difference on ASC number between the fresh and coldstored samples (Figure 1). The average of G3PDH activity was 0.22 0.02 in fresh samples, but decreased slightly in the cold-stored samples as the storage time was extended. The significant difference was found in the samples stored in refrigerator for 96 h (P < 0.05) as it compared to the fresh samples (Figure 2). No bacteria were detected in any of 10 cold-stored lipoaspirates. Comparing to long-term deep-freezing of lipoaspirate, the effect of short-term refrigeration of lipoaspirate has not been investigated extensively. Matsumoto et al.4 reported that cold-storage of lipoaspirats for 3 days reduces ASC yield, but not ASC biological properties. Eom et al.5 found that ASC population was not reduced after 36 h storage of lipoaspirates at 4 C. Our results seems more correlate to the findings from Eom et al. However, the experiment methods among these studies were different. Many factors could affect the results including patient age, sex, body mass index, medical conditions, liposuction locations and techniques, etc. However, in the present study, the influence of these factors has been largely diminished or controlled by our experimental design because all 5 portions of lipoaspirates came from the same individual. Therefore, we can make an apple-to-apple comparison. One of the unique characteristics of ASCs is that they can adhere to the plastic surface. The non-adherent cells (either non-ASCs or dead ASCs) in the flask were removed by PBS washing after 24 h of SVF culture. Our previous studies1e3 have shown that most of cells adherent to the plastic surface after 24 h SVF culture were viable (negative for both Annexin V-FITC and PI) and the ethanol-treated dead ASCs were unable to adhere to the flasks. Therefore, adherent cells from SVF culture not only can be defined as ASCs but also as viable ASCs. The controversy for the present study could be the clinical implications for using refrigerated lipoaspirate. This is the issue regarding how to deal with the remaining lipoaspirate after fat
http://dx.doi.org/10.1016/j.bjps.2014.08.070 1748-6815/ª 2014 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wang WZ, et al., The impact of short-term refrigeration of human lipoaspirate on adipose-derived stem cells and adipocytes, Journal of Plastic, Reconstructive & Aesthetic Surgery (2014), http://dx.doi.org/10.1016/j.bjps.2014.08.070
+
MODEL
2
Figure 1 storage.
Correspondence and communication
The number of adherent adipose-derived stem cell (SEM) in the lipoaspirates after 24 h, 48 h, 72 h and 96 h of cold-
Figure 2 G3PDH activity in the lipoaspirates after 24 h, 48 h, 72 h and 96 h of cold-storage. *Indicates statistically significant differences (P < 0.05) between fresh and cold-stored lipoaspirates.
grafting procedure. The unused lipoaspirate can be discarded or keep it temporarily in a refrigerator just in case you may need it in next a few days. It is rare but not impossible that an undercorrection or asymmetry in the early post graft period may be found when the patient revisits. Having readily available refrigerated lipoaspirates would be desirable for both surgeons and patients for the potential correction or secondary correction at least it is harmless. In summary, we report that human lipoaspirates stored in refrigerator for first 2e3 days after liposuction are still relatively good quality and sterile. This lays the groundwork for potential injection of refrigerated lipoaspirates. However, this result may need to be verified by an in-vivo study using refrigerated lipoaspirates.
Funding None.
Conflict of interest None.
Ethical approval The Institutional Review Board of the University of Nevada, Reno approved all of the protocols involving human tissue and cells.
Please cite this article in press as: Wang WZ, et al., The impact of short-term refrigeration of human lipoaspirate on adipose-derived stem cells and adipocytes, Journal of Plastic, Reconstructive & Aesthetic Surgery (2014), http://dx.doi.org/10.1016/j.bjps.2014.08.070
+
MODEL
Correspondence and communication
References 1. Wang WZ, Fang XH, Williams SJ, et al. Analysis for apoptosis and necrosis on adipocytes, stromal vascular fraction and adipose-derived stem cells in human lipoaspirates after liposuction. Plast Reconstr Surg 2013;131:77ce85c. 2. Wang WZ, Fang XH, Stephenson LL, et al. The effect of lipoaspirates cryopreservation on adipose-derived stem cells. Aesthet Surg J 2013;33(7):1046e55. 3. Wang WZ, Fang XH, Williams SJ, et al. Lidocaine-induced ASC apoptosis (tumescent vs. local anesthesia). Aesthet Plast Surg 2014. http://dx.doi.org/10.1007/s00266-014-0387-2. 4. Matsumoto D, Shigeura T, Sato K, et al. Influences of preservation at various temperatures on liposuction aspirates. Plast Reconstr Surg 2007;120:1510e7. 5. Eom YW, Lee JE, Yang MS, et al. Rapid isolation of adipose tissue-derived stem cells by the storage of lipoaspirates. Yonsei Med J 2011;52(6):999e1007.
3 Wei Z. Wang Xin-Hua Fang Shelley J. Williams Linda L. Stephenson Richard C. Baynosa Nolan Jaeger Kayvan T. Khiabani William A. Zamboni Department of Surgery, Division of Plastic Surgery, University of Nevada School of Medicine, Las Vegas, NV, USA E-mail address: [email protected] 14 March 2014
Please cite this article in press as: Wang WZ, et al., The impact of short-term refrigeration of human lipoaspirate on adipose-derived stem cells and adipocytes, Journal of Plastic, Reconstructive & Aesthetic Surgery (2014), http://dx.doi.org/10.1016/j.bjps.2014.08.070