Fat grafting to the face for volume augmentation, and skin rejuvenation have become a popular procedure. However, the main obstacles to fat grafting include the unpredictable volume maintenance rate and the unpredictable number of treatments needed to obtain a satisfactory rejuvenate effect. Therefore, many patients need repeat sessions. However, serial fat grafting with fresh fat imposes a burden on the patient not only because of the pain but also because of the downtime of harvesting. Therefore, if the fat can be cryopreserved, and used many times in 1 harvesting, those burdens can be reduced.
Fat grafting to the face is useful for facial rejuvenation because that procedure can return the patients’ facial volume back to the state when they were young and is effective not only for volume augmentation but also for skin rejuvenation.
Obstacles to fat grafting include the unpredictable maintenance rate for volume augmentation and the unpredictable number of treatments needed to obtain a satisfactory skin rejuvenation effect. Therefore, many patients need repeat sessions.
Serial fat grafting with fresh fat imposes a burden on the patient not only because of the pain but also because of the downtime of harvest part. If the fat can be preserved and used many times at 1 harvesting, those burdens can be reduced.
The quality of the fat after cryopreserving and thawing in the proper way is enough for clinical use. The cryopreserved fat could be a new option for serial fat grafting for facial rejuvenation.
One of the biggest reasons for an aging face is the loss of facial volume in both the bony and soft tissues. Since Coleman published the report on “structural fat grafting,” fat grafting has become popular, and many investigators confirmed the utility of fat grafting for facial rejuvenation. Fat grafting is thought to be an ideal method for facial rejuvenation because it is effective not only for volume augmentation but also for improving skin quality.
However, the main problem associated with fat grafting for volume augmentation is the unpredictable volume retention rate. Consequently, it may not be enough with only 1 operation. Therefore, serial injections are often needed to reach the ideal volume and obtain an effective outcome. In addition, fat grafting requires fat harvesting, which is painful for patients and time-consuming for surgeons. Therefore, it is ideal if the surgeon can use the fat many times in 1 harvesting.
Although many researchers have concluded that cryopreservation of fat is useful under good conditions, almost all such studies were experimental. Very few articles have described the clinical use of fat grafting with cryopreserved fat; moreover, those articles contained a very small number of patients.
In this article, the author demonstrates the outcome and ascertains the safety and efficacy of cryopreserved fat for facial rejuvenation based on 173 patients.
Special considerations for cryopreservation of the fat
The author informs the patient of the fact that the rate of maintaining fat grafting is still uncertain, so patients may need serial injections for a satisfied result. Then, the author suggests an alternative option, that the remaining fat can be cryopreserved and stored and used again without reharvesting. If the patient wishes for a serial fat injection with cryopreserved fat, the author explains that we can harvest more fat than can be used at 1 time and cryopreserve the remaining fat.
Only patients who provided written informed consent regarding postoperative infection, inflammation, oil cysts, allergy, fat necrosis, and other potential complications were included in the study. Patients aged less than 19 years and those with diabetes and severe malignant disease were excluded. Consent is obtained that the fat cannot be cryopreserved and stored for anyone who is affected with human immunodeficiency virus, hepatitis C virus, hepatitis B virus, and other severe blood infections.
In Japan, the Act on the Safety of Regenerative Medicine (Regenerative Medicine Safety Act) came into effect as of November 25, 2014 under an institutional framework for promoting the implementation of regenerative medicine. This act, which covers clinical research and private practice, stipulates 3 risk-dependent standards and the procedures for notification of plans for regenerative medicine as well as the standards of cell culture and processing facilities and the licensing procedures to ensure the safety of regenerative medicine.
In accordance with this act, CellSource Co, Ltd (Tokyo, Japan; Certification Number: FA3160006) has been certified as a cell processing center (CPC) by the Ministry of Health, Labor, and Welfare of Japan. In addition, fat grafting with the fat which is centrifuged and/or cryopreserved in a certified facility is approved in Japan ( Fig. 1 ).
Most patients were sedated by intravenous anesthesia and provided local anesthesia by a tumescent technique, but without intubation. No patient was under general anesthesia with intubation during surgery.
For fat grafting, the author collected fat by using a tumescent technique (1 mL epinephrine, 20 mL 8.4% sodium hydrogen carbonate, and 50 mL 1.0% lidocaine per 1000 mL of saline solution). The liposuction pressure was set between approximately 70 kPa and 50 kPa.
The first choices of donor sites were areas of the thighs and (lower) abdominal flank waist (so-called localized fat deposit), according to Geissler and colleagues ; the author also deems those parts as being easy to harvest and to cause less bleeding.
Injection on the Same Day of Harvesting (First Injection)
The harvested fat was usually used on the same day as tissue augmentation surgery for facial rejuvenation and/or revitalization/fertilization. In patients who underwent volume augmentation, the author applied the Coleman technique (centrifuge 1200 g for 3 minutes), and for skin texture rejuvenation (revitalization/fertilization), the author applied the nanofat technique (emulsified fat) or squeezed fat.
The detailed injection technique is in accordance with other investigators; however, the main injection parts for volume augmentation for rejuvenation were (1) forehead and temporal; (2) malar; (3) cheek; (4) nasolabial fold; (5) labiomandibular fold; (6) lips; (7) upper eyelid; (8) lower eyelid; (9) chin. And for skin texture, (1) around the lips; (2) lower eyelid; (3) wrinkles, such as between the eyebrows, forehead, and crow’s foot, at the author’s institution.
Sending the Collected Fat
The residual fat of the first injection was transferred into the transport bag (FB-bag; CellSource Co, Ltd), which contains an adipose tissue transport medium, is packed in a box called the Adiporter (CellSource Co, Ltd), and is then sent to the CPC at CellSource Co, Ltd in a refrigerated state (<10°C; Fig. 2 ).
Fat Cryopreservation and Storage
The fat-storage processing was performed in the CPC of CellSource Co, Ltd. In brief, the total fat tissue was washed with enough volume of Ringer’s lactate solution and centrifuged at 470 g for 3 minutes. The washed fat was rocked with same volume of cryoprotective solution for at least 15 minutes. After removal of the excess cryoprotectant, 4 to 5 mL of the cryoprotectant incorporated fat was transferred to cryovials. The cryovials were cooled at 1°C per minute in a controlled rate freezer to −80°C. Then, for long-term storage, cryopreserved fat was transferred to the liquid nitrogen tank and stored at −196°C. Consequently, the author can make many samples in 1 trip ( Fig. 3 ).
Regarding the process of cryopreserving and storage, in the initial stage, CellSource Co, Ltd followed procedures of the American CryoStem Corp. However, CellSource Co, Ltd has changed the protocol of processing and uses their own processing method. Concerning cryoprotective solution, they used ACSelerate-CP (American CryoStem) before, but now they use their own cryoprotective solution because they would like to improve the cell survival rate.
Recall of the Fat
After recalling the required amount of fat through the Internet Web-ordering service of CellSource, the cryopreserved fat was thawed rapidly at 37°C for 6 to 10 minutes in the CPC of CellSource. After thawing, the cryoprotective agents of the fat were washed out with flush centrifuge at 470 g . The recovered fat was filled in small syringes (usually a 5 mL syringe and containing about 4 mL fat) and sent to the author’s institution in a refrigerated state (<10°C).
Injection of Cryopreserved Fat
When the author received the fat, he injected it as soon as possible, ideally within 48 hours of being sent to the institution. As for the first injection, the author basically applied the Coleman technique, and for skin texture rejuvenation (revitalization/fertilization), he applied the nanofat (emulsified) technique. The same body parts were not necessarily injected; for example, if the first injection is for the forehead, then, for the second time, the author can inject to the chin.
The author can recall the cryopreserved fat again through the Internet service of CellSource Co, Ltd, if residual fat was present. This system allows for serial fat injections for treatment.
Additional studies for cryopreserved fat
Stromal Vascular Fraction Count
The author counted the number of stromal vascular fractions (SVF) in the cryopreserved and thawed fat before injecting into 5 patients. The SVF was isolated by washing and digested by collagenase (Wako Pure Chemical, Osaka, Japan) for 30 minutes at 37°C in a shaking water bath. Cell numbers and viability of SVF were measured with an automated cell counter (LUNA-STEM Automated Fluorescence Cell Counter; Logos Biosystems, South Korea).
Histologic Analysis of Cryopreserved Fat
The returned fat tissues obtained from the same donor were fixed with 10% formaldehyde for histologic analysis with hematoxylin and eosin (H&E) staining.
Basically, postoperative care of cryopreserved fat is the same as for fresh fat.
Namely, after fat grafting (fresh and cryopreserved), one should avoid tight compression with bandages or massages.
Regarding the next fat grafting to the same area with cryopreserved fat, one should wait at least 3 months, because the remodeling of the injected fat continues for 3 months.
For better results, a waiting period of 6 months is generally recommended to allow for initial swelling and resorption to subside.