Anesthesia

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.

Harvesting

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.

Donor Sites

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 ).

Transport materials. ( A ) FB-bag (CellSource Co, Ltd), which contains an adipose tissue transport medium. ( B ) Adiporter (CellSource Co, Ltd), which is the box kit for transportation. The fat was sent in the Adiporter to the CPC at CellSource Co, Ltd in a refrigerated state (<10°C).

( From [ A , B ] Ohashi M, Chiba A, Nakai H, et al. Serial injections of cryopreserved fat at -196°C for tissue rejuvenation, scar treatment, and volume augmentation. Plast Reconstr Surg Glob Open. 2018;18(5):e1742; with permission.)

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 ).

The collected fat is divided into 4-mL syringes at CellSource Co, Ltd as shown. Consequently, they can make many samples in 1 trip. This picture was taken after cryopreservation and thawing and returned our clinic. It looks fresh, and there is almost no oil in those syringes.

( From Ohashi M, Chiba A, Nakai H, et al. Serial injections of cryopreserved fat at -196°C for tissue rejuvenation, scar treatment, and volume augmentation. Plast Reconstr Surg Glob Open. 2018;18(5):e1742; with permission.)

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.

Repeat Injections

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.

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.