ORIGINAL ARTICLES

Cryolipolysis for Noninvasive Fat Cell Destruction:

Initial Results from a Pig Model
BRIAN ZELICKSON, MD, BARBARA M. EGBERT, MD,y JESSICA PRECIADO, PHD,z JOHN ALLISON, PHD,z
KEVIN SPRINGER, AS (ELECTRONICS TECHNOLOGY),z ROBERT W. RHOADES, PHD,y AND DIETER MANSTEIN, MDz

BACKGROUND Liposuction is one of the most frequently performed cosmetic procedures in the United
States, but its cost and downtime has led to the development of noninvasive approaches for adipose
tissue reduction.
OBJECTIVE To determine whether noninvasive controlled and selective destruction of fat cells (Cry-
olipolysis) can selectively damage subcutaneous fat without causing damage to the overlying skin or
rise in lipid levels.
METHODS Three Yucatan pigs underwent Cryolipolysis at 22 sites: 20 at cooling intensity factor (CIF)
index 24.5 (43.8 mW/cm 2), one at CIF 24.9 (44.7 mW/cm 2), and one at CIF 25.4 (45.6 mW/cm2). Treated
areas were evaluated using photography, ultrasound, and gross and microscopic pathology. Lipids were
at various times points. One additional pig underwent Cryolipolysis at various days before euthanasia.
RESULTS The treatments resulted in a significant reduction in the superficial fat layer without damage
to the overlying skin. An inflammatory response triggered by cold-induced apoptosis of adipocytes
preceded the reduction in the fat layer. Evaluation of lipids over a 3-month period following treatment
demonstrated that cholesterol and triglyceride values remained normal.
CONCLUSIONS Cryolipolysis is worthy of further study because it has been shown to significantly
decrease subcutaneous fat and change body contour without causing damage to the overlying skin and
surrounding structures or deleterious changes in blood lipids.
The research was funded by Zeltiq Aesthetics. Drs. Brian Zelickson, Barbara Egbert, and Robert Rhoades
are paid consultants to Zeltiq. Dr. Dieter Manstein receives royalty payments from Zeltiq related to licensing of
the Cryolipolysis technology. Dr. Jessica Preciado, Dr. John Allison, and Kevin Springer are employees of
Zeltiq Aesthetics.

L iposuction is a frequently performed aesthetic

operation. It has been used extensively and is
generally safe and well tolerated,1–4 but patient and
a replacement for, this procedure.5,6 Radiofrequency
methods may also be employed for removal of ad-
ipose tissue, but these techniques have been associ-
physician interest in less invasive methods to reduce ated with complications that include appearance of
body fat has resulted in significant research effort di- second-degree burns, persistent erythema, scarring,
rected toward the development of less invasive alter- edema, and fat atrophy.7 Infrared light, alone or in
native and complementary procedures for fat removal. combination with other methods, has also been em-
ployed to remove fat, with limited success.8,9 Laser-
High-intensity focused ultrasound (HIFU) has been assisted lipolysis with a medium-pulse 1,064-nm
evaluated as a noninvasive alternative for liposuction neodymium-doped yttrium aluminum garnet
but is currently considered an adjunct to, rather than (Nd:YAG) system, a new method of removing

Department of Dermatology, University of Minnesota Medical School, Minneapolis, Minnesota; yDepartment

of Dermatology and Pathology, Stanford University Medical Center, Stanford, California; zZeltiq Aesthetics,
Pleasanton, California; yRullo Communications, Steamboat Springs, Colorado; zDepartment of Dermatology, Harvard
Medical School, Boston, Massachusetts

& 2009 by the American Society for Dermatologic Surgery, Inc.  Published by Wiley Periodicals, Inc. 
ISSN: 1076-0512  Dermatol Surg 2009;35:1462–1470  DOI: 10.1111/j.1524-4725.2009.01259.x

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 ZELICKSON ET AL

localized areas of fat and tightening skin, is also Treatment

being evaluated10 but still requires tumescent
All animals were treated using a three-section cooling
anesthesia and liposuction to remove the damaged
applicator with individual cooling sections connected
fat. Additional laser systems (e.g., CoolTouch
to a prototype Zeltiq cooling control device (Figure 1)
CoolLipo 1,320, CoolTouch Corporation, Roseville,
(Zeltiq, Pleasanton, CA). To ensure consistent thermal
CA and the Palomar 920, Palomar Medical Tech-
conduction from skin to applicator, a single-use pro-
nologies, Inc., Burlington, MA) have also been
tective sleeve with nine embedded thermistors and
developed.11,12 Subcutaneous injection of phosphati-
thermal coupling gel (Figure 1) were used to couple
dylcholine and deoxycholate has also been tested for
the applicator to the skin surface in the treatment
fat dissolution.13 Although these procedures may
area. For each pig, a template was traced onto the
represent significant treatment advances, they are
area to be treated. The skin was scored with a marker
still invasive and may not provide a significant
along the top and right edges of the location where
improvement in risk–benefit ratio over liposuction.
the template contacted the body surface so the exact
Results from several studies have suggested that fat locations treated could be relocated and assessed
cells may be more sensitive to cold than other tis- during follow-up. The placement of treatment areas
sues.14,15 Results from studies exploring the effect of (Figure 2) and the treatment regimens varied some-
cold on tissue destruction have suggested that tem- what for each pig. Pigs A, B, and C received a single
peratures as high as 11C can decrease the viability of treatment 90 days before euthanasia, and pig D was
adipocytes.16–18 All of these findings suggest that treated at multiple sites 90, 60, 30, 14, 7, and 3 days
application of cold to the skin may provide a non- and immediately (30 minutes) before euthanasia.
invasive approach to fat removal. Approximately 25% to 30% of the total body surface
area was treated in each animal.
The objective of the present study was to test
noninvasive cold exposure regimens to determine Pig A was treated at three sites with the rate
whether selective damage to subcutaneous fat can be of energy extraction set to a CIF value of 24.5
induced without damage to the overlying skin and (43.8 mW/cm2). For each of the treated areas, the
without causing a harmful rise in lipid levels. cooling applicator extracted heat from the underly-
ing tissue for 60 minutes. A mechanical vibration of
the cooling applicator was used to massage the tissue
Methods

Experimental Animals

The study was conducted in three Yucatan pigs (A, B,

and C; 24–30 months of age, body weight approx-
imately 115 kg) and one Yorkshire pig (D; 96 kg).

Determination of Energy Withdrawal

The cooling intensity factor (CIF) setting on the

proprietary research device is a numerical value that
regulates a heat extraction (cooling) rate that trans-
lates to milliwatts per centimeter squared (mW/cm2).
The higher the CIF, the more rapid the rate of energy
extraction. The other major treatment variable was
time over which the research device, set to a chosen Figure 1. Photograph of two prototype Zeltiq cooling appli-
CIF value, was applied to the skin surface. cators being used to treat Pig A.

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A 24.5. Each site was treated for 45 minutes, including a

5-minute period of tissue massage. Pig D was treated
at 20 sites with the CIF set at 21.5 (36.8 mW/cm2).
Each site was treated for 15 minutes.

Assessments

Treated and adjacent areas were evaluated using

standardized flash photography and diagnostic ultra-
sound (SonoSite 180 [SonoSite Inc., Bothell, WA]
with a 7.5-MHz linear transducer) assessments 3
B
months after treatment. At the time of necropsy, tissue
was collected for gross pathologic and standard
histologic evaluation. Histological sections were
stained with hematoxylin and eosin and evaluated
microscopically to assess the level of fat damage and
any damage to the dermis or epidermis. Lipid panels
were performed for each animal using blood samples
collected after a 12-hour fast before treatment and 1
day, 1 week, and 1, 2, and 3 months after treatment.

C
Results

Evidence of Efficacy

Visual inspection of the Yucatan pigs 3 months after

treatment revealed noticeable smooth inward con-
tour changes on the surface of the treated areas
(Figure 3). These contour changes correlated to de-
creased thickness of the fat layer as measured using
ultrasound, with the greatest reductions measured in
tissue areas that received the more intense and longer
Figure 2. Treatment layouts for pigs (A–C). Each rectangle duration treatment. The changes in surface contour
represents one treatment site. Each animal was treated on shown in Figure 3 reflect a substantial decrease in
only one side of its body to produce a region of large surface
adipose tissue underlying the contoured area. The
area that extended over a uniform fat layer.
reduction in thickness of the upper layer of the
gently for 5 minutes during each cooling treatment. subcutaneous fat is demonstrated in the gross pa-
Pig B underwent cooling treatment at eight sites thology photographs shown in Figure 4.
(Figure 2, Pig B, A–H) using a cooling applicator and
a prototype cooling control device. The rate of energy Reduction in the fat layer thickness for each animal
extraction for this pig was set to a CIF value of 24.5. was assessed using ultrasound (Figure 5) and mea-
Sites A and B were treated for 60 minutes, including a surement of pathologic specimens. Results for Pig A
5-minute period of massage. Each of the other sites indicated a reduction in the superficial fat layer of
was treated for 45 minutes, including a 5-minute pe- 33% (from 2.1 to 1.4 cm); those for Pig B indicated a
riod of massage. Each site on pig C was treated with reduction of 33% (from 1.8 to 1.2 cm). These data
the rate of energy withdrawal set to a CIF value of were not collected for pig C.

1464 D E R M AT O L O G I C S U R G E RY
 ZELICKSON ET AL

Figure 3. Photographs of treatment areas from two animals showing skin surface contour changes 3 months after treatment.

Examination of specimens collected for gross patho- ment. Between 14 and 30 days after treatment,
logic analysis (Figure 6) also demonstrated reductions phagocytosis of lipids is apparent. By 30 days after
in superficial fat in treated areas. Results from one pig treatment, the inflammatory process had begun to
indicated a decrease in the thickness of this layer of decline, and by 60 days, it appears that the thickness
53% (from 1.9 to 0.9 cm); those from a second pig of interlobular septa has increased. The inflamma-
indicated a decrease of 50% (from 2.0 to 1.0 cm). tory process declined further by 90 days after treat-
These data were not collected for the third pig. ment, and the increase in the thickness of septa was
also pronounced at that time. This is believed to be
Histologic analysis of tissues taken from Pig 4 dem- the result of selective removal of adipocytes, reduc-
onstrated that Cryolipolysis resulted in the death of ing the thickness of the tissue and thereby increasing
adipocytes that macrophages subsequently engulfed the proportion of collagen in the adipose tissue. Also
and digested (Figure 7). The progression of the in- apparent in the sequenced photomicrographs in
flammatory process that resulted in the phagocytosis Figure 8 is the substantial loss of fat cells in the
of lipids is illustrated in Figure 8. Immediately after treated area. Histologic analysis indicated no dis-
treatment, there are no changes in subcutaneous fat. cernable damage to the dermis or epidermis in any
By 3 days after treatment, there is evidence that an of the areas treated. There was no ulceration or
inflammatory process stimulated by adipocyte ap- necrosis of the epidermis or dermis. In addition,
optosis has begun, as reflected by an influx of in- no necrosis was observed in appendageal
flammatory cells. This inflammatory process became structures, such as hair follicles or sweat glands
increasingly apparent at 7 and 14 days after treat- (Figure 9).

Erythema was observed immediately after treatment

and resolved within 30 minutes. The skin was
cold to the touch, although not hard or icy, after
treatment. There was no evidence of edema,
bruising, purpura, or scarring at the time of any
follow-up examination or on the day of necropsy.

Changes in Lipids

Assessment of lipids over the 3-month follow-up

period demonstrated some variation over time, but
Figure 4. Gross pathology photograph showing an example
of fat layer reduction responsible for the visible skin contour these small changes remained within the bounds of
changes shown in Figure 3. normal reported for these animals19 (Table 1).

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Figure 5. Ultrasound measurements of fat layer thickness demonstrating reductions in the superficial fat layer for two of the
three Yucatan pigs included in this study. In each pair of images, that on the left was taken before treatment, and that on the
right was taken 3 months after treatment.

Discussion body contour and reductions in the superficial fat

layer of pigs. Although the cold was applied to the
Results from this preliminary study indicate that
skin surface, the damage was confined to the fat, and
Cryolipolysis induced using controlled exposure to
no damage to the epidermis or dermis could be
cold can induce selective damage to the subcutane-
found. Evaluation of lipids at multiple time points
ous fatty tissue, resulting in subsequent changes in
after treatment indicated that lipids remained within
the bounds of normal variation.

Evaluation of histologic specimens collected after

treatment indicated that the reduced thickness of
the fat layer was associated with an inflammatory

Figure 6. Gross pathology sections showing reductions in Figure 7. The natural inflammatory response to cold expo-
superficial fat layer 90 days after treatment. sure in one area treated.

1466 D E R M AT O L O G I C S U R G E RY
 ZELICKSON ET AL

Figure 8. Progression of inflammatory response to cold exposure in tissue taken from pig D: (A) 3 days, (B) 7 days, (C) 14
days, (D) 30 days, (E) 60 days, (F) 90 days.

Figure 9. Tissue section from the epidermis and dermis of the same treatment site shown in Figure 7 (tissue taken 90 days
after treatment).

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TABLE 1. Posttreatment Changes in Lipids from selective loss of fat cells without damage to the
Baseline in Pigs A to C overlying skin and associated structures may reflect
the fact that adipocytes are more sensitive to cold
Posttreatment Day than other cell types.14,15 Reperfusion of adipose
Lipid 7 28 59 89 tissue rendered ischemic by cold exposure may result
in oxidative stress, including elevation of lipid per-
Cholesterol
A 11.6 16.3 20.9 16.3 oxidation and reduced glutathione levels, ultimately
B 7.7 23.1 0 9.9 resulting in cell death.25,26
C 5.6 5.6 0 7.4
Low-density lipoprotein cholesterol
A 16.7 72.2 50.0 50.0
These results suggest that adipocyte apoptosis pro-
B 25.8 51.6 0 16.1 vided the stimulus for the observed inflammatory
C 41.2 17.6 0 11.8 response, although it is likely that this response also
High-density lipoprotein cholesterol contributes to the total number of adipocytes killed.
A 40.0 30.0 5.0 5.0
B 2.9 20.0 0 2.9 The inflammatory response observed appeared sim-
C 7.7 7.7 0 30.8 ilar to that reported previously in subcutaneous fat
Triglycerides after exposure to intense cold.27 As noted in the In-
A 22.2 14.8 3.7 33.3
troduction, exposure of fat cells to cold in situations
B 55.9 5.5 0 52.8
C 53.7 16.7 0 51.8 in which an inflammatory response is not likely (e.g.,
storage of isolated cells) results in adipocyte
Days for evaluation in Pig C were 8, 29, 59, and 90.
death.16,17 These findings also support the view that
the observed inflammatory response is a conse-
response. This response peaked approximately 1 quence rather than a cause of adipocyte death.
month after treatment and then declined. Although
the inflammatory response had clearly declined by 3 Study results indicated that application of cold did
months after treatment, the modest residual inflam- not result in any damage to the epidermis or dermis.
mation at the time when the pigs were euthanized The reason for the lack of effect of cold exposure on
suggests that greater decreases in the fat layer may skin or associated structures is most probably the
have been observed with longer survival times. carefully controlled condition under which heat is
Studies in which animals survive for longer periods being extracted from the adipose tissueFthe proto-
after treatment are needed to determine the full time type device used in this study regulates the rate
courses of the inflammatory processes and remod- of energy extraction from the skin (measured in
eling that follow Cryolipolysis. Two mechanisms of mW/cm2) through monitoring of several electronic
fat cell loss have been described in the literature thermistor sensors on the skin surface. In other sit-
(dedifferentiation and apoptosis),20 and the results uations, cold exposure can result in significant in-
presented are consistent with the conclusion that flammation of the skin,28–30 but fat appears to be
exposure to cold induces apoptosis of fat cells. Re- more susceptible to cold exposure and to ischemia
view of the literature related to cryosurgery, a much and ischemia-reperfusion injury than other tissues,
more aggressive procedure than Cryolipolysis, indi- including the skin.25,31
cates that cell death associated with cryosurgery
represents apoptosis rather than necrosis,21,22 and it Cryolipolysis relies on natural thermal diffusion to
seems reasonable to suggest that apoptosis is also the realize a gradual and tapered effect within the fat
mechanism underlying fat cell death in the present layer. Controlling the rate of energy extraction and
study. The extraction of heat from adipose tissue duration of treatment may be used to limit the
may also set the stage for ischemia-reperfusion injury amount of tissue subject to Cryolipolysis according
that has been shown to result in apoptosis.23,24 The to treatment goals.

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 ZELICKSON ET AL

The use of Cryolipolysis for reducing the superficial 9. Sadick N, Magro C. A study evaluating the safety and efficacy of
the VelaSmooth system in the treatment of cellulite. J Cosmet
fat layer may have cosmetic applications in humans, Laser Ther 2007;9:15–20.
although it is not known whether the levels of cold
10. Katz B, McBean J. The new laser liposuction for men. Dermatol
exposure necessary for Cryolipolysis pose a unique Ther 2007;20:448–51.
risk to patients with rare conditions such as cry- 11. Dierickx CC, Khatri KA, Tannous ZS, et al. Micro-fractional
oglobulinemia, paroxysmal cold hemoglobinuria, or ablative skin resurfacing with two novel erbium laser systems.
Lasers Surg Med 2008;40:113–23.
cold urticaria. Future study of Cryolipolysis in hu-
12. Yang CH, Chou HS, Lo YF. Incompetent great saphenous
mans should address these potential risks.
veins treated with endovenous 1,320-nm laser: results for
71 legs and morphologic evolvement study. Dermatol Surg
In conclusion, Cryolipolysis, a new method of con- 2006;32:1453–7.

trolled energy extraction (cooling) from adipose tis- 13. Schuller-Petrovic S, Wölkart G, Höfler G, et al. Tissue-toxic
effects of phosphatidylcholine/deoxycholate after subcutaneous
sue, has been evaluated as a means of predictably injection for fat dissolution in rats and a human volunteer.
destroying adipocytes while preserving the skin and Dermatol Surg 2008;34:529–43.

surrounding structures. This noninvasive procedure 14. Wiandrowski TP, Marshman G. Subcutaneous fat necrosis of the
newborn following hypothermia and complicated by pain and
achieves fat loss through heat extraction applied for
hypercalcaemia. Australas J Dermatol 2001;42:207–10.
an extended period of time. The results indicate that
15. Diamantis S, Bastek T, Groben P, Morrell D. Subcutaneous fat
Cryolipolysis treatment merits further study because necrosis in a newborn following icebag application for treatment
it leads to significant changes in body contour and of supraventricular tachycardia. J Perinatol 2006;26:518–20.

can decrease subcutaneous fat without damaging the 16. Lidagoster MI, Cinelli PB, Leveé EM, Sian CS. Comparison of
autologous fat transfer in fresh, refrigerated, and frozen speci-
overlying skin or causing harmful changes in blood mens: an animal model. Ann Plast Surg 2000;44:512–5.
lipids.
17. Wolter TP, von Heimburg D, Stoffels I, et al. Cryopreservation of
mature human adipocytes: in vitro measurement of viability. Ann
Plast Surg 2005;55:408–13.

18. Manstein D, Laubach H, Watanabe K, Farinelli W, Zurakowski

References D, Anderson RR. Selective cryolysis: a nivel method of non-
invasive fat removal. Lasers Surg Med 2008;40:595–604.
1. Heymans O, Castus P, Grandjean FX, Van Zele D. Liposuction:
review of the techniques, innovations and applications. Acta Chir 19. Rispat G, Slaoui M, Weber D, et al. Haematological and plasma
Belg 2006;106:647–53. biochemical values for healthy Yucatan micropigs. Lab Anim
1993;27:368–73.
2. Stewart KJ, Stewart DA, Coghlan B, et al. Complications of 278
consecutive abdominoplasties. J Plast Reconstr Aesthet Surg 20. Avram MM, Avram AS, James WD. Subcutaneous fat in normal
2006;59:1152–5. and diseased states: 1. Introduction. J Am Acad Dermatol
2005;53:663–70.
3. Yoho RA, Romaine JJ, O’Neil D. Review of the liposuction,
abdominoplasty, and face-lift mortality and morbidity risk liter- 21. Baust JG, Gage AA. The molecular basis of cryosurgery. BJU Int
ature. Dermatol Surg 2005;31:733–43. 2005;95:1187–91.

4. Coldiron BM, Healy C, Bene NI. Office surgery incidents: what 22. Yang WL, Addona T, Nair DG, et al. Apoptosis induced by cryo-
seven years of Florida data show us. Dermatol Surg 2008;34:285– injury in human colorectal cancer cells is associated with mi-
91; discussion 291–2. tochondrial dysfunction. Int J Cancer 2003;103:360–9.

5. Rohrich RJ, Beran SJ, Kenkel JM, et al. Extending the role of 23. Rentsch M, Beham A, Iesalnieks I, et al. Impact of prolonged cold
liposuction in body contouring with ultrasound-assisted liposuc- ischemia and reperfusion on apoptosis, activation of caspase 3,
tion. Plast Reconstr Surg 1998;101:1090–102. and expression of bax after liver transplantation in the rat.
Transplant Proc 2001;33:850–1.
6. Pine JL, Smith LJ, Haws MJ, Gingrass MK. Ultrasound-assisted
lipoplasty. Plast Surg Nurs 2003;23:101–8. 24. Tanaka M, Mokhtari GK, Terry RD, et al. Prolonged cold
ischemia in rat cardiac allografts promotes ischemia-reperfusion
7. de Felipe I, Del Cueto SR, Pérez E, Redondo P. Adverse reactions injury and the development of graft coronary artery disease
after nonablative radiofrequency: follow-up of 290 patients. in a linear fashion. J Heart Lung Transplant 2005;24:
J Cosmet Dermatol 2007;6:163–6. 1906–14.

8. Nootheti PK, Magpantay A, Yosowitz G, et al. A single center, 25. Coban YK, Kurutas EB, Ciralik H. Ischemia-reperfusion injury of
randomized, comparative, prospective clinical study to determine adipofascial tissue: an experimental study evaluating early histo-
the efficacy of the VelaSmooth system versus the Triactive system logic and biochemical alterations in rats. Mediators Inflamm
for the treatment of cellulite. Lasers Surg Med 2006;38:908–12. 2005;2005:304–8.

35:10:OCTOBER 2009 1469

 C RY O L I P O LY S I S F O R N O N I N VA S I V E FAT C E L L D E S T R U C T I O N

26. Werker PM, Kon M, Green CJ, et al. Oxidative processes and free 30. de Souza RC, Cunha JM, Ferreira SH, et al. Different inflamma-
radical scavengers in ischaemia-reperfusion injury in adipocuta- tory mediators induce inflammation and pain after application of
neous flaps: in vitro lipid peroxidation assessment. Br J Plast Surg liquid nitrogen to the skin. Cryobiology 2006;53:319–29.
1995;48:590–6.
31. Donski PK, Franklin JD, Hurley JV, O’Brien BM. The effects of
27. Ter Poorten JC, Hebert AA, Ilkiw R. Cold panniculitis in a ne- cooling on experimental free flap survival. Br J Plast Surg
onate. J Am Acad Dermatol 1995;33:383–5. 1980;33:353–60.

28. Page EH, Shear NH. Temperature-dependent skin disorders. J Am

Acad Dermatol 1988;18:1003–19.

29. Ozyazgan I, Tercan M, Melli M, et al. Eicosanoids and inflam-

matory cells in frostbitten tissue: prostacyclin, thromboxane, Address correspondence and reprint requests to: Brian
polymorphonuclear leukocytes, and mast cells. Plast Reconstr Zelickson, MD, 4100 West 50th St, Edina MN 55424,
Surg 1998;101:1881–6. or e-mail: [email protected]

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