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Title:
Cell-Assisted Lipotransfer for Breast Augmentation: Grafting of Progenitorenriched Fat Tissue

Authors:
Kotaro Yoshimura, M.D.,1 Katsujiro Sato, M.D.,2 Daisuke Matsumoto, M.D.1

Affiliations:1

Department of Plastic Surgery, University of Tokyo School of Medicine;

7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
2

Cellport Clinic Yokohama;

Yokohama Excellent III Building 2F, 3-35, Minami-nakadori, Naka-ku, Yokohama,
Kanagawa 231-0006, Japan

Corresponding author:
Kotaro Yoshimura, M.D.
Department of Plastic Surgery, University of Tokyo School of Medicine;
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
Telephone: +81-3-5800-8948; Fax: +81-3-5800-8947
E-mail: yoshimura-pla@h.u-tokyo.ac.jp

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Abstract
Lipoinjection is a promising treatment, but is currently limited by unpredictable outcomes
and a low rate of graft survival due to partial necrosis. To address these problems wedeveloped a novel strategy called Cell-Assisted Lipotransfer (CAL) in which autologous
adipose-derived stem (stromal) cell (ASC) supplementation is used in combination withlipoinjection. A stromal vascular fraction (SVF) containing ASCs is isolated from half ofan aspirated fat sample and is recombined with the remaining half of the aspirated fat
sample. This process converts relatively progenitor-poor aspirated fat to progenitor-rich fat.
Our experience with the CAL technique showed that by transplanting the ASC-enriched
fat tissue post-operative atrophy of transplanted fat grafts was minimal and satisfactory
clinical results were generally achieved without any major complications, suggesting that
ASC supplementation is both effective and safe. Further studies with longer follow up arenecessary to establish the value of this technique. Continued improvements in thetechnique could make autologous tissue transfer the first choice for breast augmentation in
the future.

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I. Introduction
Autologous fat transplantation is a promising cosmetic treatment for facial rejuvenationand soft-tissue augmentation because of the lack of an incision scar and complications
associated with foreign materials. However, certain problems remain, including
unpredictable outcomes and a low rate of graft survival due to partial necrosis. Autologous
fat transplantation has been used for breast augmentation by only a limited number ofplastic surgeons [1]; this procedure is controversial due to the lack of consensus onwhether it is safe and appropriate because of associated microcalcifications that mightcause confusion during the evaluation of mammograms. Recently, autologous fat injection
has been re-evaluated as a potential alternative to artificial implants for breast
augmentation [1-5]. This re-evaluation may reflect recent advances in autologous fat
transfer and the radiological detection of breast cancer.
In this chapter, we introduce a novel approach to autologous fat grafting called cellassisted lipotransfer (CAL). CAL is the concurrent transplantation of aspirated fat tissue
and adipose progenitor cells called adipose-derived stem/stromal cells (ASCs), which is
the grafting of progenitor-enriched fat tissue (Fig. 1). The therapeutic strategy is based on
the observation that aspirated fat is vessel-poor and adipose progenitor cell-poor ascompared to intact whole fat [6].

II. Adipose tissue-specific progenitors with multipotency called adipose-derived
stem/stromal cells
It has been shown that fibroblast-like stromal cells obtained from liposuction aspirates candifferentiate into various cell lineages [7,8] such as adipogenic, osteogenic, chondrogenic,

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myogenic, cardiomyogenic, and neurogenic. Thus, the adipose tissue-specific progenitor
cells are now called “adipose-derived stem/stromal cells (ASCs)” and are expected to
become valuable tools in a wide range of cell-based therapies [9] (Fig. 2). Adipose tissue
is known to be rich in microvasculature [10], and ASCs were shown to have angiogenic
characteristics and to experimentally differentiate into vascular endothelial cells [6,11,12].
Human ASCs are distinct from other mesenchymal progenitors in their surface marker
expression profile; notably, only ASCs express stem-cell–associated marker CD34 inhigher percentages than do bone-marrow–derived mesenchymal stem cells and dermal
fibroblasts [8].
ASCs are currently being used in clinical trials of treatments for bone defect(autologous fresh ASCs) [13], rectovaginal fistula (autologous cultured ASCs) [14], graftversus-host disease (non-autologous ASCs) [15], and soft tissue augmentation by CAL(autologous fresh ASCs) [5]. If ASCs are harvested from a large volume (e.g., 500 ml) of
liposuction aspirates, ASCs can be used without cell expansion because a sufficientnumber can be obtained from such a volume. Furthermore, the use of minimallymanipulated fresh cells might lead to higher safety and efficacy in actual treatments.

III. Biological and therapeutic concepts of cell-assisted lipotransfer
1)

Cell components of adipose tissue Adipose tissue consists predominantly of

adipocytes, ASCs, endothelial cells, pericytes, fibroblasts, and extracellular matrix.
Adipocytes constitute more than 90% of tissue volume but they are much larger in size
than the other cells and the number of adipocytes is estimated to be only about 50-60% of
the total cell number[16] (Fig. 3). ASCs are adipose tissue-specific progenitor cells that5

contribute to adipose tissue turnover (adipose tissue is considered to turnover every 2-3
years [17]) and provide cells for the next generation. Based on recent studies, ASCs are
considered to be bipotent progenitors, being sources of both adipogenic and angiogenic
lineages [12].

2)Aspirated fat versus intact fat In general, we can use only aspirated fat tissue aslipoinjection material. Aspirated fat is a fragile part of the adipose tissue taken undernegative pressure. Indeed, a fibrous honeycomb structure is left in the donor tissue after
liposuction. Our research revealed that aspirated fat contains only half the number ofASCs as intact whole fat [6] (Fig. 4). The two main reasons for this relative deficiency ofASCs are 1) a major portion of ASCs are located around large vessels (within tunica
adventitia) and are left at the donor site, and 2) part of ASCs are released into the fluid
portion of liposuction aspirates [8]. Our histological studies indicated that ASCs arelocated not only between adipocytes but also around vessels. Large-sized vessels arelocated in the fibrous part of the tissue contained by intact whole fat but much less by
aspirated fat. Thus, aspirated fat tissue is regarded as progenitor-poor fat tissue ascompared to intact fat tissue.

3)

Stromal vascular fraction Through collagenase digestion, a heterogeneous cell

mixture can be extracted from adipose tissue as a cell pellet. This cell fraction is called the“stromal vascular fraction (SVF)” (Fig. 2) because it contains mostly stromal cells,
vascular endothelial cells, and mural cells, but not adipocytes. In the clinical setting, theSVF contains a substantial amount of blood-derived cells such as leucocytes and6erythrocytes as well as adipose-derived cells such as ASCs and vascular endothelial cells.
Our pilot study [16] revealed that nucleated cells contained in SVF are composed of 37%
white blood cells, 35% ASCs, and 15% endothelial cells and other cells, though thepercentage of blood-derived cells strongly depends on individual hemorrhage volumes. In
CAL, the freshly isolated autologous SVF is used to supplement fat graft tissue without
any manipulations such as cell sorting or culture.

4)

Progenitor:adipocyte ratio In general, tissue grafting is performed using graft

tissue with an intact organ-specific ratio of progenitor cells:differentiated adult cells. For
example, in split- or full-thickness skin grafting, the graft skin has the same number ofbasal keratinocytes and other keratinocytes as intact skin has. The ratio of basal
keratinocyte number to other differentiated cell number is the progenitor:mature-cell ratio
for the epidermis.
In adipose tissue, aspirated fat has a significantly lower progenitor:mature-cell ratio, as
mentioned above, and this low ASC:adipocyte ratio might be the main reason for longterm atrophy of transplanted adipose tissue. There are at least three experimental studies,including ours [6,18,19], demonstrating that supplementing adipose progenitor cells
enhances the volume or weight of survived adipose tissue.
We have found that centrifugation of the aspirated fat influences engraftment
efficiency substantially, because centrifugation at 1,200×g decreases the fat volume by
30%, damaging 12% of the adipocytes and 0% of the ASCs [20] (Fig. 5). This leads tocondensation of cell numbers per volume of adipocytes and ASCs by 25% and 43%,

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respectively, and improved the ASC/adipocyte ratio by 14%. Thus, even centrifugation
alone is likely to lead to better aspirated fat engraftment.

5)

Concept of Cell-Assisted Lipotransfer: Enrichment of adipose progenitor cells

by supplementation with the stromal vascular fraction Supplementation with SVF
improves the progenitor/adipocyte ratio − progenitor-poor aspirated fat tissue is converted
to progenitor-rich fat tissue. It was hypothesized that this progenitor-enriched fat tissue
would not only survive better but would also preserve its volume with minimal atrophy. In
CAL, freshly isolated SVF, which contains ASCs, is added to progenitor-poor aspirated fat
tissue; the cells are attached to the aspirated fat before transplantation with the fat acting as
a living bioscaffold (Fig. 1). After transplantation, the ASC-supplemented adipose tissue
encounters ischemia and subsequent reperfusion, the high pressure of edema, andinflammatory changes in the host tissue. The microenvironments, injury-associated growth
factors, and inflammation-associated cytokines and chemokines influence ASC behavior
during the acute phase of tissue repair, as discussed in the next paragraph.

6)

Possible roles of adipose-derived stem/stromal cells in Cell-Assisted

Lipotransfer There are four possible roles for ASCs in CAL, which have partly
confirmed in pre-clinical studies [6,18,19]. First, ASCs differentiate into adipocytes andcontribute to the regeneration of adipose tissue. Second, ASCs differentiate into
endothelial cells and possibly vascular mural cells [6,11,12], thereby promoting
angiogenesis and graft survival. Third, ASCs release angiogenic growth factors such as

8hepatocyte growth factor in response to injury, hypoxia, and other conditions [21, ourunpublished data], and these factors influence surrounding host tissue. Finally, andpossibly most importantly, some ASCs survive as original ASCs [6]. In the adipose, ASCs
reside between adipocytes or in the extracellular matrix, especially around vessels, andcontribute to the turnover of adipose tissue, which is known to be very slow (2 years ormore) [17]. However, surviving adipose grafts probably turn over during the first 2 to 3
months after transplantation because they experience temporary ischemia followed byreperfusion injury. This turnover, the replacement process of the adipose tissue, isconducted by tissue-specific progenitor cells, which are ASCs. The relative deficiency ofASCs in aspirated fat could affect the replacement process and lead to post-operative
atrophy of grafted fat, which commonly occurs during the first 6 months following
lipoinjection.

IV. Technique1)

Surgical procedures Donor sites are determined according to patient’s preference

and body mass index (BMI). If the patient’s BMI is greater than 25 then 1,500 ml ofaspirated fat can usually be harvested from either the abdomen and flanks, or posterior,
medial, and lateral thighs. If BMI is less than 20, fat should be harvested from both theabdomen and thighs. After the liposuction site is infiltrated with saline solution containing
diluted epinephrine (0.001%) under general anesthesia, the adipose tissue is suctioned
using a cannula with 2.5-mm inner diameter and a conventional liposuction machine.
About a half of the collected liposuction aspirate (500-800 ml of aspirated fat) is used toharvest the SVF. The SVF is isolated from both the adipose portion and the fluid portion

9of liposuction aspirates, as described below [8], and the cell processing procedure takes
about 80 min. During the processing period, the remaining half of lipoaspirate is harvested
and prepared as a graft material. The aspirate is centrifuged at 700×g for 3 min and thefloating adipose portion is placed in a metal jar (1,000 ml) that is placed in an ice waterbath.
For the injection syringe, we use a 10 cc LeVeen™ inflator (Boston Scientific Corp.,MA) or our original syringe (20 ml) because they are both screw-type syringes (with athreaded plunger) with threaded connections that fit both the connecting tube and theneedle, providing precise control during injection (Fig. 6). We use two syringes in order toreduce the time of the procedure; while one syringe is being used for an injection the otheris filled with the graft material in preparation for the next injection. A 16 or 18-gauge
needle (150-mm long) is used for lipoinjection and inserted subcutaneously at one of thefour points indicated in Figure 7A. Care is taken to insert the needle horizontally (parallelto the body) in order to avoid damaging the pleura and causing a pneumothorax. The
needle is inserted in several layers and directions and is continuously and gradually
retracted as the plunger is advanced (Fig. 7B), thereby ensuring diffuse distribution of thegraft material. The grafts are placed into the fatty layers on, around, and under the
mammary glands (but not intentionally into the mammary glands), as well as into thepectoralis muscles. After training, the operator can easily recognize the difference between
mammary gland or pectoralis fascia, which are harder tissues, and the fat or muscle tissue.
After the surgery, the breasts are maintained in the proper position using a brassier;massage of the breasts is prohibited during the first three months.

10For patients with artificial breast implants, CAL can be performed immediately
following implant removal. During the cell isolation period, the breast implants are
removed through a periareolar incision made at the caudal third of the areola margin.
Lipoinjection is initiated at the deepest layer under the implant capsule and completedwith injection into the most superficial subcutaneous layer. Again, in the deepest layer, it
is important to insert and place the needle horizontally (parallel to the body) in order toavoid damaging the pleura. The operator can insert a finger into the implant capsule andplace it on the bottom of the capsule to recognize the location of the injection needle. The
needle is inserted from the lateral margin of the breast and from a point on the
inflamammary fold. Lipoinjection between the capsule and the skin is done from the sametwo points and from the periareolar incision. This technique helps to ensure a diffuse
distribution of the graft material; no injections are made into the mammary glands or into
the capsular cavity. Finally, the capsular cavity is washed with saline and the periareolar
incision is closed.

2)

Cell processing (stromal vascular fraction isolation procedure) Processedlipoaspirate cells (PLA) cells and liposuction aspirate fluid (LAF) cells are separated fromthe fatty and fluid portions of liposuction aspirates, respectively. For PLA cells, thesuctioned fat is digested with 0.075% collagenase in phosphate buffered saline for 30 min
on a shaker at 37°C after centrifugation. Mature adipocytes and connective tissues are
separated from cell pellets by centrifugation (800×g, 10 min), the pellets are thenresuspended in erythrocyte lysis buffer (155 mM NH4Cl, 10 mM, KHCO3, 0.1 mM
EDTA) and incubated for 5 min at room temperature. The pellets are resuspended and11passed through a 100-mm mesh filter (Millipore, Billerica, MA). To eliminate any
remaining collagenase, the cells pellets are washed at least three times in Dulbecco’s
Modified Eagle’s Medium (DMEM) by repeated suspension and centrifugation. For LAF
cells, the suctioned fluid is centrifuged (400×g, 10 min) and the pellets resuspended inerythrocyte lysis buffer. After 5 min at room temperature, lysates are passed through a
100-mm mesh filter. Again, the cell pellets are washed at least three times in DMEM andpassed through a 100-mm mesh filter.
The entire procedure should be performed by well-trained physicians or technicians in
an aseptic room (preferably at a level of good manufacturing practice) according to a
designated standard operating procedure. Isolated cells should be strictly evaluatedregarding quantity and quality. Cell counts for erythrocytes and nucleated cells are
performed using a cell counter used for standard blood testing. The whole process of cellisolation takes about 70-80 minutes. We also recommend that a fraction of the isolated
SVF be seeded and cultured to verify cell viability and another fraction be frozen andstored in a deep freezer or liquid nitrogen for future cell tracing.

V. Results of clinical trials
1)

Patients From 2003 to 2008, we performed CAL in 188 patients at various

anatomical sites, including 164 breast procedures (20 patients had breast reconstruction
after mastectomy), 37 facial procedures, two procedures in the hand, and three in the hip(CAL was performed at two different sites in 17 patients). In 164 breast cases, 26 patients
underwent CAL immediately after removal of breast implants. All of the patients werefemales with a BMI of 19.6 ± 2.1 (mean ± standard deviation) and the patient’s ages

12varied from 13 to 73 years (34.9 ± 11.2). The mean volume of injected fat was 268.6 ±48.2 ml on the left side and 273.1 ± 40.4 ml on the right.

2)Pre- and post-operative evaluations In order to evaluate outcomes, we took

physical measurements (maximum and bottom breast circumferences, etc.), and performed
mammography, magnetic resonance imaging (MRI), echography, photography, andvideography. We have also adopted a three-dimensional (3D) measurement system thatenables volumetric evaluation of the breast mound in a standing position. Perpendicularstriped lights are projected onto the breasts and photographed using a stereo-type digital
camera (Fig. 8). The digital images are then analyzed using customized software. We
calculate the volume and projection of each breast above a standard plane designated bythree fixed points (the shoulder, suprasternal notch, and xiphoid process) that do not
usually shift after breast augmentation.

3)

Outcomes The procedure takes about 3.5-4 hours including SVF isolation. The

injection process requires 35-60 min for both breasts. Subcutaneous bleeding and edema
are typical on some parts of the breasts, but this usually resolves within one to two weeks.
Transplanted adipose tissue was gradually absorbed during the first two postoperative
months, particularly during the first month, and the breast volume changed minimallythereafter, although skin tension sometimes decreased after two months. The 3D
measurements taken at six months follow up showed that the surviving fat volume was100-250 ml, meaning that the graft take ranged from 40-90% (Fig. 9). Compared to breasts
augmented with implants of the same size, breasts augmented with CAL were lower but

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had a more natural contour and softness without any palpable nodules at six months follow
up. Patients were satisfied with the outcome despite the limited size increase possible withautologous tissue transfer. Computed tomography (CT) scans and MRI showed thattransplanted fat tissue survived and formed a significant thickness of the fatty layer not
only subcutaneously on and around the mammary glands but also between the mammary
glands and the pectoralis muscles.
Regarding CAL-mediated breast augmentation immediately after implant removal,
most cases showed natural softness of the breasts without any palpable nodules at six
months follow-up, and the patients were satisfied with the texture, softness, contour,
symmetry, and the absence of foreign material.
These satisfactory outcomes are similar to those observed in other soft tissue
augmentation cases, such as in patients with hemifacial lipoatrophy [22].

4)Representative cases Two representative cases of breast augmentation by CAL

and two cases of breast augmentation by CAL immediately after implant removal areillustrated in Figures 10-13.

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VI. Discussion
1)

Refinement of autologous fat graft techniques It is well accepted that adipose

tissue should be grafted in small aliquots, preferably within an area 3-mm in diameter [23].
Because it requires a substantial length of time to perform ideal, diffuse distribution ofsuctioned fat into the breast [1], we use a disposable syringe with a threaded plunger andconnections and a very long needle (150 mm). These devices are critical for performing
large-volume lipoinjection safely and precisely in the shortest length of time possible. We
use a relatively large-sized suction cannula (2.5-3.5-mm inner diameter), centrifuge theaspirated fat, and keep it cooled until transplantation. In our experience, outcomes(increase in breast size) are superior when centrifuged versus non-centrifuged fat is used,although we have yet to perform a quantitative and statistical analysis of this observation.
The reason that centrifuged fat produces better outcomes could be that the ASC:adipocyte
ratio is improved following centrifugation [20]. In addition, centrifugation may be ofparticular benefit in this procedure because centrifugation decreases the water content in
the graft material. Higher water content could disturb the ASCs to adhere to the adipose
tissue, leading to unexpected behaviors of ASCs, as discussed below.
After transplantation, ASCs probably interacts with other cells contained in SVF
such as vascular endothelial cells. Therefore, in this treatment, supplementation with the
SVF might be superior to supplementation with ASCs alone. Further studies are needed toelucidate the synergistic effects of ASCs with other cells contained in the graft.

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2)

Indications There are several patient factors that may affect the clinical outcomeof CAL, such as skin redundancy of the breasts, age, BMI, personal quality or character offat, scars and adhesions, breast implant and its capsule, systemic disease such as
autoimmune disease, and oral corticosteroid use. Lean patients have a disadvantage
because it is not easy to obtain 1,500 cc of fat from these patients. Some lean young
patients with no history of pregnancy have flat chests and high skin tension, therefore theycannot accept a large volume fat graft due to skin shortage. Some patients have oily
aspirates and others have fibrous aspirates. Mastectomy patients have scarring andadhesions to the underlying fascia and some have a history of radiation therapy.
Good candidates for CAL are those who have sufficient fat at the donor sites and sufficientskin redundancy on breasts with healthy skin vasculatity and no scars. In our experience,
age does not appear to affect the clinical result.
Patients with breast implants, who are already familiar with drawbacks of implants andhave sufficient breast skin expanded by implants, are considered good candidates for CAL
even though they have implant capsules in place. Similarly, the breast skin of women witha history of pregnancy and breast feeding has expanded due to enlargement of the
mammary glands and their breasts can more easily accept a larger injection volume thanthose with no history of pregnancy.

3)Complications Cyst formation (5-15 mm diameter) was detected by MRI in twopatients and by echogram in six patients. Tiny cyst formation (smaller than 5 mm) only
detected by echogram might happen more frequently, but no treatment is needed as long as
the cyst diameter is less than 10 mm. Small calcifications were detected by mammogram

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in two patients at 24 months follow up, but the calcifications were easily distinguishedfrom those associated with breast cancer. Postoperative donor site problems, such as
irregularity or seroma, could be more commonly associated with CAL than withconventional treatment because of the large volumes removed during liposuction.
In two patients in which an SVF cell suspension was injected into each breast mound(30 ml per side) immediately after conventional lipoinjection, the breast mounds were
somewhat hard to the touch at three months; CT scan detected unexpected fibrosis in the
subcutaneous fat layers of the breast mounds and fibrosis on the sternum [24]. Therefore,
ASCs should be adhered to cells, tissue, extracellular matrix, or some type of biologicalscaffold prior to administration in order to avoid their unexpected differentiation,
migration, or other behavior.

VII. Conclusions
Transplanting ASC-enriched fat tissue provided satisfactory outcomes without any majorcomplications. Our experiences with the CAL technique suggest that ASC
supplementation is a safe and effective means of breast augmentation. Controlled studies
with longer follow-up are necessary to establish the value of this technique. Continued
improvements to this technique could make autologous tissue transfer the first choice forbreast augmentation in the future.

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Legends

Fig. 1. Scheme of Cell-Assisted Lipotransfer (CAL). Relatively adipose-derived
stromal/stem cells (ASC)-poor aspirated fat is converted to ASC-rich fat by
supplementation with ASCs isolated from half of the aspirated fat sample. The ASCs areattached to the aspirated fat, which is used as a scaffold in this strategy (cited from ref#22). SVF, stromal vascular fraction.

Fig. 2. The stromal vascular fraction (SVF) can be obtained from adipose and fluid
portions of liposuction aspirates through collagenase digestion. SVF contains 10-35%
adipose-derived stromal cells (ASCs), some of which are multipotent and have beenshown to differentiate into several lineages in vitro. SVF also contains blood-derived cells
such as leukocytes.

Fig. 3. Scheme of adipose tissue components. Adipocytes constitute more than 90% of
tissue volume but only 50-60% of the total cell number. Adipose-derived stromal/stem
cells (ASCs), endothelial cells, fibroblasts, and other cells constitute the remainder.
Extracellular matrix (ECM) of the adipose tissue contains various collagens, laminin,
fibrinogen, and other ECM substances.

Fig. 4. Comparison of human aspirated fat and excised whole fat obtained from a single
site of a single patient. (top) Histology of aspirated fat and excised fat. (hematoxylin andeosin-stained microphotographs and scanning electron micrographs; red scale bar = 200

23μm, white scale bar = 40 μm). The basic structure of adipose tissue was preserved in theaspirated fat while significantly fewer vascular vessels, especially those of large size, weredetected in aspirated fat than in excised fat. It is well known that the honeycomb structuresof vascular and neural perforator networks are left intact in aspirated sites following
liposuction procedures. (bottom) Adipose-derived stromal/stem cell (ASC) yield fromaspirated fat and excised fat. Both tissues were processed for isolation of stromal vascular
fractions, which were then cultured for one week. Ratios of ASC yields from aspirated fat
to ASC yields from excised fat of the same volume were calculated; data from threepatients (#1–#4) and their average value are shown. The ASC yield from aspirated fat wassignificantly less (56 ± 12 %) than the yield from excised fat (cited and revised from ref.
#6).

Fig. 5. Effects of centrifugation on aspirated adipose tissue. The adipose portion wasconcentrated to 71.0% of the original volume after centrifugation for three minutes at
1200×g. The volume of the adipose portion was significantly reduced and the volume of
the fluid and oil portions was significantly increased. However, the number of adiposederived stromal cells (ASCs) contained in the adipose portion was not significantly
changed by the centrifugation. Thus, centrifugation at 1,200×g led to condensation of cellnumbers per volume of adipocytes and ASCs by 25% and 43%, respectively, andimproved the ASC:adipocyte ratio by 14% (cited and revised from #19).

Fig. 6. Injection devices. A high-pressure injection can be performed with a disposable
syringe with a threaded plunger. A 150-mm long 16- or 18-gauge needle is connected to24

the syringe with a connecting tube threaded at both ends. The injection needle is rigidly
manipulated by an operator, while an assistant rotates the plunger according to theoperator’s instruction.

Fig. 7. Schematic instruction of the injection method. (A) The needle is inserted fromeither one of two points on the areola margin or one of two points at the infra-mammary
fold in various directions and planes to achieve a diffuse distribution. (B) A small amountof fat tissue is injected in small aliquots or a thin string with a long needle on a syringewith a threaded plunger while the needle is continuously withdrawn (cited and revised
from ref. #5).

Fig. 8. Three-dimensional system for measuring breast volume. Using this system, breast
volume can be measured while the patient is in a sitting position.

Fig. 9. Sequential volume changes after cell-assisted lipotransfer measured using the threedimensional system (preliminary results for 28 patients). Augmented volume among
patients varied between 100 and 250 ml at six months, corresponding to 40-90% survivalof transplanted adipose tissue.

Fig. 10. Case 1 (breast augmentation): Preoperative views (top) and postoperative(bottom) views at 24 months. A 30 year-old woman underwent breast augmentation with
CAL (310 ml in each breast). Her breasts were augmented dramatically with an 8.0-cm
increase in breast circumference at 24 months. The breast mounds were soft with no25

subcutaneous indurations. An original infra-mammary fold on the left breast is slightly
visible, but injection scars are not visible (cited and revised from ref. #5).

Fig. 11. Case 2 (breast augmentation): Preoperative view (top) and postoperative (bottom)
view at 12 months. A 36 year-old woman whose body mass index was 17.3 underwent
breast augmentation with CAL (245 ml in each breast). The breast mounds were soft withno subcutaneous indurations or visible scars at 12 months.

Fig. 12. Case 3 (breast augmentation immediately after implant removal): A 33 year-old
woman who had 210 ml saline implants underwent implant removal and simultaneous
CAL (260 ml in each breast). The preoperative view showed capsular contractures andupward displacement of the left implant (top). At 12 months the breasts were symmetricand had a natural appearance (bottom). MRI at 12 months revealed that transplanted
adipose tissues had survived and formed thick layers around and under the mammary
gland. Mammograms showed no calcifications or other abnormal signs in either breast at
12 months. Augmented breast mounds maintained a sufficient breast volume even after
implant removal and were naturally soft without any subcutaneous indurations.

Fig. 13 Case 4 (breast augmentation immediately after implant removal): A 25 year-old
woman who had 165 ml hydrogel implants (ruptured) underwent implant removal andsimultaneous CAL (260 ml in each breast). The preoperative view showed capsularcontractures and displacement of the right implant (top). At 12 months the breasts weresymmetric and had a natural appearance (bottom), and mammography revealed no26

abnormalities. Augmented breast mounds were soft and natural appearing without
injection scars or subcutaneous indurations.

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PhotosFigure 1.

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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Figure 9

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Figure 10

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Figure 11

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Figure 12

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Figure 13