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ADIPOCYTE VIABILITY STUDY IN AN ACCREDITED CRYOLAB. EFFECT OF AGE, HARVESTING TECHNIQUE, SAMPLE SIZE, FREEZING DELAY, FREEZING DURATION, AND SHIPPING.

Bejjani3, F.J., MD, PhD; Stern1, Frederic, MD; Goldfarb2, Richard, MD; Troell Robert J, MD; Goldman3, Ruth; Moscatello3, David, PhD.

1The Stern Center for Aesthetic Surgery, Bellevue, WA; 2Center for Smartlipo, Langhorne, PA; 3American Cryostem, LLC; Advanced Body Sculpting, Las Vegas, NV

ABSTRACT In order to assess adipocyte viability through cryopreservation, and test all the SOPs of an accredited FDA registered cryolaboratory in comparison with prevailing literature, 15 consecutive samples were studied with regard to washed v. received weight, delay before freezing, duration of freezing, post-thaw viability and post-shipping (Fedex) viability. The samples came from 12 different physicians, 6 had harvested the fat manually, 6 with assistive devices and 3 undetermined. There was only one male and the mean donor subjects age was 55.13 ± 10.17 years. Two counts of percentage of live cells and cell diameters were obtained with a cellometer after both AO and PI stains. Median values were recorded and the lowest (representing the worst scenario) were used for further statistical analysis. SPSS MANOVA was utilized. Adipocyte viability was in the high 80th low 90th percentile throughout, slightly better with PI stain and slightly higher in post-thaw (probably because of mitoses resuming which is a good sign of viability). Age, sample size, delay pre-freeze, freeze duration, harvesting technique showed no statistically significant effect on adipocyte viability. Cell diameter was significantly smaller post-cryopreservation and that is thought to be due to the protective effect of the cryoprotectant glycerol solution. This study shows better viability than the literature, thus validating the SOPs and procedures utilized in the cryolaboratory in question. Further studies will now utilize these processes for SVF, adipocyte stem cells and differentiated adipocytes, in light of the numerous clinical applications in progress.

 

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ADIPOCYTE VIABILITY STUDY IN AN ACCREDITED CRYOLAB. EFFECT OF AGE, HARVESTING TECHNIQUE, SAMPLE SIZE, FREEZING DELAY, FREEZING DURATION, AND SHIPPING.

Bejjani3, F.J., MD, PhD; Stern1, Frederic, MD; Goldfarb2, Richard, MD; Troell Robert J, MD; Goldman3, Ruth; Moscatello3, David, PhD.

1The Stern Center for Aesthetic Surgery, Bellevue, WA; 2Center for Smartlipo, Langhorne, PA; 3American Cryostem, LLC; Advanced Body Sculpting, Las Vegas, NV

INTRODUCTION

The use of fat grafts for soft tissue augmentation was first described in 1893 by Neuber. Recently, riding the wave of the “heel thyself” movement and the “everything organic and natural” concerns, there has been a growing interest in the use of lipoaspirate for autologous fat transfer (AFT) and as a source material for adipose-derived regenerative cells. AFT is performed for both cosmetic and reconstructive purposes by an increasing number of physicians to provide lasting, natural structural and contour changes postoperatively, as evidenced by an increasing number of articles and presentations on this topic (Saint-Cyr et al, 2012; Bucky & Percec, 2008; Coleman, 2001; Matsumoto et al., 2006; Yoshimura et al., 2008; Bejjani et al., 2011). According to the International Society of Aesthetic Plastic Surgery, fat grafting represented 5.9 percent of the nonsurgical procedures within aesthetic surgery, with more than more than 84,000 patients treated in the United States and 514,000 procedures performed globally in 2009.

Although patient safety is the primary concern (Gutowski et al, 2009), the

ultimate retention of grafted material is also an important measure of efficacy for both patients and surgeons. Graft retention is dependent on a number of factors, many of which are still not completely understood or quantified (Smith et al, 2006; Carpaneda, 1996; Coleman, 2006). The ultimate success of the procedure depends on elements of the entire process: aspiration, processing/handling, centrifugation (Botti et al., 2011; Ferarro et al., 2011; Pulsfort et al., 2011), storage/ cryopreservation and ultimately reinjection. A number of authors have examined the effects of different approaches on each step in the process (Tommaso et al, 2012; Locke et al.,

2008; Sommer & Sattler, 2000). Examples abound in the plastic surgery literature for the use of cryopreservation technology in long-term preservation of skin grafts, as in skin grafting (Catagnoli et al., 2003) and nipples for autologous reconstruction (Nakagawa et al, 2003).

Adipose tissue, when digested with collagenase and centrifuged to remove differentiated adipocytes floating in the aqueous phase, forms a cellular pellet made of a highly

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heterogeneous population of cells, typically referred to as the stromal vascular fraction (SVF) and includes colony-forming unit-fibroblasts (CFU-fs), vascular/endothelial cells, erythrocytes, and other hematopoietic cells. These SVF can be freshly used for therapeutic applications (Yoshimua et al., 2008; Muller et al, 2010). The isolation yield of SVF cells not only depends on how the tissue is processed, but also on various factors, including interdonor variability, anatomical harvest site, aspiration procedure, storage time, sample processing prior to digestion (e.g., washing or concentration by centrifugation), and con centration/type of enzyme used. Examples can range between 4.104

cells/ml (Aust et al., 2004) and 3.105

cells/ml (Mitchell et al., 2006). The reported frequency of CFU- in SVF also varies in the literature, ranges from 0.3% to 15%, likely due to different clonal densities of seeding, different media used, or a discrepancy in the definition of a CFU-f colony (Fraser et al., 2006; Muller et al., 2010). SVF can also be seeded onto tissue culture plastic in order to select the adherent population and then expanded to generate adipose derived mesenchymal stem cells (ASCs). The use of these ASCs for cell-based therapy to reconstruct either single or composite tissue loss represents a promising and innovative option for many clinical problems (Katz et al., 1999; Ashjian et al., 2002; Hedrick et al., 2003; DeUgarte et al., 2003; Muller et al. 2010 ). Human adipose aspirates contain multilineage and multipotent cells and are an excellent source of human stem cells (Ashjian et al., 2003).

Several studies demonstrate that processed lipo-aspirate cells can differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors (Zuk et al. 2002; Zuk et al., 2001; Mizuno et al., 2002; Huang et al, 2002; Huang et al., 2004).

It appears to be more attractive to many patients to have their adipose aspirates preserved initially so that they may have an option later to undergo future repeated autologous fat transplantations for cosmetic or reconstructive soft-tissue augmentation, or any other treatments (Pu et al, 2004; Pu et al., 2006). Since the early 21st century, Pu and colleagues (University of Kentucky) have been studying the long-term preservation of autologous fatty tissues collected from conventional liposuction. They found that adipose tissue can be preserved and stored successfully at low temperature (below – 85°C) by means of an optimal cryopreservation technique. Bone Marrow v. Adipose Tissue MSCs Human bone marrow has historically been identified as a primary source of adult stem cells, primarily because of its therapeutic use in leukemia. However, traditional bone marrow harvesting procedures may be traumatic, frequently requiring anesthesia, and may yield low numbers of mesenchymal stem cells on processing (Zuk et al. 2002; Zuk et al., 2001). Adipose tissue may represent a better source of adult human stem cells because of abundant sources, similarities with bone marrow in term of embryonic origin, and easy availability

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through a liposuction procedure (DeUgarte et al., 2003).

New research has shown the presence of microangiopathy in the bone marrow of diabetic patients for the first time. The starvation of bone marrow as a consequence of microangiopathy can lead to a less efficient healing in diabetic patients. Bone marrow stem cells have been the most used in regenerative medicine trials to mend hearts damaged by heart attacks. Results from this study highlight an important deficit in stem cells and supporting microenvironment that can reduce bone marrow stem cells' therapeutic potential in diabetic patients (Spinetti et al., 2013). This bone marrow microangiopathy could explain the poor angiogenesis encountered after hematopoietic stem cell autograft for a group of lower limb ischemia patients, many of whom being diabetic (Gomez et al., 2012).

Also recently, a team of stem cell specialists and infectious disease researchers from the Forsyth Institute in Cambridge, Stanford University School of Medicine, and India unveiled preliminary evidence of a major global health mystery and an enormous problem indeed: Mycobacterium tuberculosis (Mtb) may maintain long-term intracellular viability in a human bone marrow (BM)–derived CD271+/CD45− mesenchymal stem cell (BM-MSC) population. Viable Mtb was detected in CD271+/ CD45− BM-MSCs isolated from individuals who had successfully completed months of anti-Mtb drug treatment. These results suggest that CD271+ BM-MSCs may provide a long-term protective intracellular niche in the host in which dormant Mtb can reside. Not only is this strong evidence that the tuberculosis can remain dormant in BM-MSCs, but it shows that the living bacteria

could be recovered from these cells after a long period of time. These stem cells are known to migrate to sites of injury or inflammation and begin dividing. So it is forseable that migrating stem cells harbouring dormant bacteria might reactivate the disease in the lung. This study suggests it is possible that a host of other infectious diseases may use a similar "wolf-in-stem-cell-clothing" tactic to hide away from therapies in the bone marrow (Bikul et al., 2013).

Pendleton’s et al. (2013) study from the Johns Hopkins Departments of Neurosurgery and Oncology is crucial to the future of cell therapy in many different ways: 1) it compares autologous to allogeneic: 2) it compares bone marrow MSCs to adipose; 3) it assesses their tropism towards Glioma, a very common brain tumor. Major findings and conclusions can be summarized as follows:

- Adipose-(both autologous and

allogeneic) and bone marrow-derived (allogeneic) mesenchymal stem cells appear to have similar glioma tumor tropism in vitro. The actual treatment of gliomas requires a population of cells with a doubling time and proliferation rate rapid enough to allow timely expansion of autologous cells for clinical applications.

- Pluripotentiality for both AMSC and

BMSC lines was confirmed through differentiation along three mesenchymal lineages: adipocytes, osteocytes, and chondrocytes. Commercial animal media were used (rabbit, goat, bovine, etc.) instead of human-based.

- While the volume of bone marrow that

can be harvested under local anesthesia

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brings about 2.4x104 MSCs, the volume of adipose that can be harvested this way brings about a minimum of 106 MSCs. This two-hundred-fold difference in harvest cell density makes adipose tissue the more efficient cell source. A higher seeding density is necessary for the successful growth and expansion of BMSCs, which demonstrate slower growth characteristics.

- AMSCs have a faster proliferation rate

that is retained through multiple passages. This rate is important not only in maximizing clinical applications, but also in minimizing the risk of malignant transformation, which has been linked to the length of ex vivo culture.

- Unmodified human AMSCs remain free

of oncogenic transformation for at least eight months, when injected into immune-compromised mice, demonstrating more oncogenic resistance than BMSCs.

- The BMSC population has been shown

to decrease substantially with age and diabetes casting further doubt on the use of autologous BMSCs as a therapeutic delivery vehicle in the patient population most often afflicted with gliomas. High viral counts have also been encountered in bone marrow.

- The study showed inter-patient stemness variability in the primary AMSC lines which would most likely be worse for autologous BMSC lines as described above. Hence the importance of storing one’s adipose cells while young and healthy to get the best stemness, same as recommended by cosmetic and plastic surgeons performing repeat fat grafting (Pu et al,

2004; Pu et al., 2006).

Purpose of this study

American Cryostem (CRYO) laboratory has been using a proprietary self-contained fat collection and shipping product called Atgraft for the last year or so. The purpose of this study is to measure adipocyte viability throughout the cryopreservation process, using the numerous CRYO validated and FDA registered SOPs, from the time of the Atgraft sample reception in the lab, as shipped from a doctor’s office to the time it is shipped back to that office. The goal being to provide the physicians and their patients with the most viable fat sample possible for fat transfer procedures or any other clinical implementations as the need arises.

REVIEW of LITERATURE Rate, Duration and Temperatures of Cryopreservation In the late 20th century, Armitage and others noted increased tissue viability using cryopreservative and controlled-rate freezing with storage in liquid nitrogen (Armitage, 1978; Bank & Brockband, 1987; Billings & May, 1989; Henry et al., 1993; ; Karlsson & Toner, 1996). In 2005, Wolter et al. stated that the widely used practice of simple storage in a freezer leads to reinjection of nonviable tissue. Aspirated fat should be transplanted as quickly as possible if it is preserved at room temperature. For adipose-derived stem cell isolation, aspirated fat can be stored or transported overnight if it is preserved at 4° C without adipose-derived stem cell yield loss or changes in biological properties (Matsumoto et al. 2007). A temperature of +4° C is an

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effective and easily available way of storing fat grafts for at least 2 weeks (Erdim et al., 2008). Fat preservation storage techniques using a -15° C freezer or a -70° C deep freezer are both inadequate to maintain the viability of fat cells (Son et al., 2010). Viability of adipocytes frozen at -20° C was very low when analyzed by staining, and no cultures could be established from any of the specimens. In contrast, viable adipocytes were recovered from samples that were controlled-rate frozen in the presence of cryo-protectants and stored in nitrogen vapor (Lidagoster et al., 2000; Moscatello et al., 2005). The viability of adipose aspirates frozen with dimethyl sulfoxide at -80° C was approximately 87% after 2 months of storage. Moreover, ASCs from adipose tissue stored with cryoprotectant survived successfully for 1 year and differentiated into adipocytes (Lee et al., 2010). The cryopreserved adipose tissue, once it became equilibrated in liquid nitrogen in approximately 20 minutes, is considered to be equivalent to one after long-term preservation and was ready for thawing. (Pu et al., 2006). Pu, Gao, Moscatello and others also emphasized the importance of controlled-rate freezing and thawing, i.e. slow freezing fast warming. ASCs can be long-term cryopreserved, and this, due to their great numbers, is an attractive tool for clinical applications (DeRosa et al., 2009; Pu, 2009).

Cryoprotective Agents

More maintained volume, weight, and fatty tissue structure of injected free grafts are found in the optimal cryopreservation

group (with cryopreservant) compared with the simple cryopreservation group, but the results were still less satisfactory than those in the fresh control group (Pu et al., 2006).

In The Prospect of Immortality, the book

which launched cryonics, Robert C.W. Ettinger (1964) suggests that glycerol might be used as the cryoprotectant for human cryopreservation based largely on the fact that it was the dominant cryoprotective agent (CPA) at that time (1962-64) and most of the positive results with sperm and tissues had been achieved with glycerol. Due largely to the flare and flamboyance of “the Father of Dimethyl Sulfoxide (DMSO),” Dr. Stanley W. Jacob, who was Assistant Professor of Surgery at the University of Oregon, DMSO entered the public consciousness in a big way in the mid-to-late 1960s (Jacob et al., 1971). DMSO’s antiinflammatory, and seemingly incredible skin-penetrating properties, were much talked about.

As per the round-table conference held at the National Academy of Science in 1976 (US NCIRR, FDA & NMRI) the consensus was that in general, the classical penetrating cryoprotective agent, glycerol, has been used. The recovery of live spermatozoa after freezing in various concentrations of glycerol has been reported by several investigators. Optimum concentations ranged between 5 and 10% (Matheson et al. 1969; Pedersen & Lebech, 1971; Perloff et al, 1964; Rubin et al., 1969; Swada, 1964; Sherman, 1962), Other researchers compared 0, 5, 10, and 15% glycerol and obtained respective recovery rates of 13, 72, 46 and 15% (Freund, 1971). Karow (1972) compared glycerol with dimethylsulfoxide (DMSO) and reported that 5-10% of either compound is protective. DMSO and glycerol were reported as equal at 10%

(Sherman, 1964); while one study showed that glycerol was superior to DMSO as a

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cryoprotectant for human spermatozoa (Zimmerman et al., 1964). More recently, Moscatello found that the DMSO group showed 64% viability as compared to 50% for the glycerol group (Moscatello et al., 2007). The combination of 0.5M Me(2)SO and 0.2M trehalose as CPA in addition to the controlled slow cooling and fast rewarming protocol, appears to provide the maximum recovered results in cryopreservation of human adipose tissues (Cui et al., 2007). Trehalose, serving as a CPA in its optimal concentration, appears to provide adequate protection of human fat grafts during cryopreservation in vivo (Pu et al., 2005) Such protection is similar to that provided by the combination of DMSO and trehalose as a CPA. Because of its safety and effectiveness, trehalose can possibly be administered to patients for long-term preservation of their fat grafts (Cui & Pu, 2009; Cui & Pu, 2010). One of the leaders of cryobiology and cryopreservation, Pr Dayong Gao of University of Washington (Gao et al, 1997; Baust et al., 2009) performed extensive research with his team to determine the optimal cryoprotectants (Gilmore et al., 1997). He found that glycerol reduced thermal stress during cell freezing (Lin & Gao, 1991); and eliminated the fracture phenomena in an isotonic salt solution during freezing (Gao, Lin et al., 1995). He also studied the prevention of osmotic injury to sperm during addition and removal of glycerol (Gao, Liu et al., 1995). Karlsson and Toner (1996) selected dimethyl sulfoxide, a permeable cryoprotective agent that could reduce cell injury caused by the intracellular ice formation and “solution effects,” and trehalose, a nonpermeable cryoprotective agent that could protect the cell membrane. For freezing of

Markhoz goat sperm, glycerol was found to be a better CPA than DMSO (Farshad, et al., 2009).

Viability Stains A landmark turn-of-the-century study revealed uniformly poor scientific methodology in assessing adipocyte viability. Nine of 16 studies used no histologic evaluation of grafts in their studies, and two additional studies did not examine reinjected fat graft survival (Sommer & Sattler, 2000). Shoshani and his team examined fat grafts by standard hematoxylin-eosin staining 15 weeks after subcutaneous injection into the scalp region of immunodeficient mice (Shoshani et al., 2001) . Several researchers used a dual live-dead staining method fluorescein diacetate and propidium iodide, which stain viable and nonviable cells, respectively (Jones & Senft, 1985; Moscatello et al., 2007). Acridine orange (AO), Hoechst 33342 (HO) and propidium iodide (PI) are among the most used fluorescent dyes used to analyze cell culture viability. In fact, they respectively show specificity for living, apoptotic and late apoptosis/necrosis states (Foglieni et al. 2001). The AO/PI viability assay is a rapid, highly linear, functionally correlated assay that is superior to conventional viability measurement (Mascotti et al., 2000). The AO/PI staining procedure allows for the identification of viable, early membrane-intact apoptotic and necrotic cell populations (Simpson et al., 1997; Al-Rubeai & Fassenegger, 2004). Trypan blue staining was also used to determine the vitality of defrosted adipocytes (Sommer & Sattler, 2000).

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Post-thaw Viability, Culture and Volume

Moscatello et al. (2007) utilized an alternative method to determine the viability of the mature adipocytes and adipose stromal cells (ASCs), which is a slight modification of the Sugihara’s ceiling culture method (Sugihara et al., 1986; Sugihara et al., 1987): The washed fat was added to 15% FBS in DMEM:HF12 (Ham’s nutrient mixture F12) (1:1) plus 10 µg/mL gentamicin in a Nunc T25 flask, and more medium was added to completely fill the flask, avoiding. The flasks were tightly capped and transferred to a tissue culture incubator “bottom” up. Because the mature adipocytes float, viable adipocytes would adhere to the upper surface of the flasks, whereas the ASCs would settle and adhere to the bottom surface. In post-thaw ceiling culture, the slower cell growth in the cryopreservation group in comparison with the control group may also represent an insignificant mechanical injury to processed lipoaspirate cells within adipose aspirates during the process of obtaining specimens used in this study (Pu et al., 2005). Because cell viability stains are very difficult to quantitate on clumps of adipose tissue, a standard collagenase digestion technique is routinely used to dissociate the recovered adipose tissue (Strutt & Killinger, 1996; Moscatello et al., 2007). Shoshani et al. reported no significant differences in the volumes or histology of the grafts between the fresh and frozen recipients, there was a significant reduction in volume (to approximately 60%) in both

graft classes (Shoshani et al., 2001) Adipose Stem Cells (ASCs), after being thawed, are still capable of differentiation and express all surface antigens detected before storage, confirming the integrity of their biology. In particular, ASCs differentiated into adipocytes, showed diffuse positivity for PPARgamma and adiponectin, and were also able to differentiate into endothelial cells without addition of angiogenic factors (Billings & May, 1989; DeRosa et al., 2009). Manual v. Assisted Harvesting

As described by Coleman, fat harvested specifically for grafting is gently aspirated by handheld syringes with small blunt cannulae (Coleman, 2006).

Autologous fat grafts harvested with the Coleman technique (Coleman, 2001). and preserved with Dr Pu’s lab cryopreservation method have a normal histology with near the same number of viable adipocytes as compared with the fresh fat grafts. However, those cryopreserved fat grafts appear to have a less optimal level of adipocyte specific enzyme activity compared with the fresh ones and thus may not survive well after they are transplanted without being optimized (Pu et al., 2010). Fat cells survive aspiration with a suction machine or syringe equally well. Use of a liposuction cannula or 14-gauge needle gives comparable results (Sommer & Sattler, 2000.) The use of larger liposuction cannulas for fat tissue harvesting provides more viable fat grafts (Erdim et al., 2008).

Adipose tissue acquired via third=generation UAL (VASER; Sound Surgical Technologies, Inc, Louisville, Colorado) is viable at harvest

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and is potentially a suitable source for autologous fat grafts. These results confirm reported clinical successes utilizing third-generation ultrasound lipoaspirate for AFT (Schafer et al., 2013)

As compared with the manual process, Sepax automation resulted in a 62% higher isolation yield, with 2.6–1.2·105 nucleated ASCs cells per mL of liposuction, and a 24% higher frequency of clonogenic progenitors. The variability in the isolation yield and clonogenicity across different preparations was reduced by 18% and 50%, respectively. The cytofluorimetric profile and in vitro differentiation capacity into mesenchymal lineages were comparable in the cells isolated using the two procedures (Guven et al, 2012).

In a recent study out of Massachusetts

General, higher aspiration pressures up to −0.83 atm did not affect fat graft viability in vivo. Positive pressure up to 6 atm also did not affect fat graft viability. The degree of shear stress, which is a function of flow rate, did significantly affect fat graft viability. Fat grafts injected slowly with low shear stress significantly outperformed fat injected with high shear stress (Lee et al. 2013).

MATERIALS

Fifteen consecutive fat samples that were received at the CRYO lab between 3/13/13 and 9/19/13 were included in this study, all from female donors except one. The samples were collected by 12 different physicians nationwide, Six physicians used the manual harvesting kit provided with the Atgraft, 6 used power-assisted devices and 3 did not specify. Physicians had contacted the lab to receive the Atgraft kit with return shipping materials and tracking numbers as per the CRYO SOP.

Upon receiving the returned sample, the fat is weighed and washed following another validated SOP and all characteristics were charted. After initial staining with both Acridine Orange (AO) and Propidium Iodide (PI), and prefreeze viability measurement, cryopreservant was applied, i.e glycerol 10%. The samples were then slowly brought to deep freeze, following the corresponding SOP. After a variable number of days (6 to 206), the samples were thawed, staining and viability measurements were repeated as in prefreeze above. Finally from the thawed fat of three donors, two samples were created for each, a bag (25 cc) and a tube (5 cc) and they were both shipped via Fedex, as if being sent back to the contributing physician, only the recipient in these cases was the CRYO lab itself. Upon receipt of the shipped samples, they were all submitted to repeated viability measurements as above. Analysis Table 1 lists the descriptive statistics

such as range, minimum, maximum, mean and standard deviation that were obtained for all independent variables: age of the donors, days lapsed before cryopreservation was initiated, received fat weight, washed fat weight, supernate (ratio between the latter two), and duration of cryopreservation prior to repeating the viability analysis. Using the Nexcelom Cellometer, two viability counts per specimen were performed, after both Acridine Orange (AO) and Propidium Iodide (PI) stains, following the Nexcelom procedure and the CRYO SOP. The median viability percentages were charted for each count, as well as the median adipocyte diameters. For the following statistical analyses only the lowest of the two viability percentages

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and the smallest of the two diameters were utilized, thus representing a worst case scenario for the purposes of this study.

Table 1: Independent Variables Descriptive Statistics

N R Min Max Mean

Std.

Dev. Age (yrs)

15

35

35

70

55.13

10.17

Received Fat (g) Washed Fat (g) Super-nate (%)

14

14

14

203

169

56

40

30

48

242

199

1.04

103.03

80.97

78.91

66.33

55.84

16.74

Prefreeze Days 15 3 1 4 1.73 .96

Freeze Duration (Days)

15 200 6 206 80.07 70.07

Median prefreeze viability numbers were found to be borderline higher for the PI stain than the AO stain (paired t-test=2.07; p=.06). Statistical comparison was performed on these numbers for manual v. power-assisted technique (Figure 1). The difference was minimal and not statistically significant. Figure 1: Adipocyte Viability (%): Comparison by Harvesting Technique

Median viability numbers were then compared between prefreeze, postfreeze and postshipping for both the AO stain (Figure 2) and the PI stain (Figure 3, Table 2) Table 2: Adipocyte Viability (%) prefreeze, postfreeze and postshipping

VIABILITY (%) N R Min Max Mean

Std.

Dev.

Pre-freeze (AO)

15

48.6

50.7

99.3

82.53

14.09

Post-freeze (AO)

11 22 76 98 87.75 7.73

Post-ship (AO)

3 10.3 84.7 95.0 90.13 5.17

Pre-freeze (PI)

15

27.2

72.8

100.0

90.94

6.67

Post-freeze (PI)

12 22 77 98 90.18 7.36

Post-ship (PI)

3 35.00 60.1

0 95.10 76.36 17.63

AO = Acridine Orange PI = Propidium Iodide

The Multiple Analysis of variance procedure of SPSS (MANOVA) was used to compare the means of theses viability measurements and gauge the effect on them of all the independent variables listed in table 1. First there was no statistically significant difference between the prefreeze, postfreeze and postshipping viability. Second, age, freezing delay or duration and ratio of washed over recieved fat, did not have a statistically significant effect on the viability.

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Figure 2: Adipocyte Viability comparison prefreeze postfreeze and post-shipping, on Cellometry after Acridine Orange (AO) Stain

Figure 3: Adipocyte Viability comparison prefreeze, postfreeze and postshipping, on Cellometry after Propidium Iodide (PI) Stain

Median adipocyte diameters were also compared between prefreeze, postfreeze and postshipping for both the the AO stain (Figure 4) and the PI stain (Figure 5, Table 3). The Multiple Analysis of variance procedure of SPSS (MANOVA) was used to compare the means of theses diameter measurements and gauge the effect on them of all the independent variables listed in table 1.

Figure 4: Adipocyte Diameter Comparison prefreeze postfreeze and postshipping, on Cellometry after Acridine Orange (AO) Stain

The postfreeze diameter was significantly smaller than the prefreeze for both the AO (p=.001) and the PI (p = .017), but then did not change further with shipping. Same as with viability measurements, age, freezing delay or duration and ratio of washed over recieved fat, did not have a statistically significant effect on the adipocytes median diameters for both stains. Figure 5: Adipocyte Diameter comparison prefreeze, postfreeze and postshipping, on Cellometry after Propidium Iodide (PI) Stain

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Table 3: Adipocyte Diameter (microns) prefreeze, postfreeze and postshipping

DIAMETER (microns) N R Min Max Mean

Std

Dev

Pre-freeze (AO)

15 90.70 27.71 118.41 89.27 20.3

Post-freeze (AO)

14 86 23 110 50.89 30.16

Post-ship (AO)

3 3.23 24.87 28.10 26.93 1.79

Pre-freeze (PI)

15

66.74

37.13

103.87

85.85

18.66

Post-freeze (PI)

13 88 36 124 60.76 27.98

Post-ship (PI)

3 4.92 35.31 40.23 38.02 2.50

AO = Acridine Orange PI = Propidium Iodide Finally in order to determine the effect of sample size on postshipping adipocyte viability and diameter, measurements obtained from both the bag and tube samples were compared for both these variables (Table 4). There was no statistically significant difference in median adipocyte viability or median diameter between the large sample (bag) and the small tube. DISCUSSION First we should remember that these viability percentages are the worst scenario, meaning the lowestr median values were taken from two different counts for each of the tests. Nonetheless values have been in

the upper 80th to 90th percentile throughout which is quite satisfactory and an improvement compared to the literature, namely Moscatello et al. They seem to be slightly higher for the Propidium Iodine than the Acridine Orange but not significantly. Figure 6: Adipocyte Viability post-shipping comparison between bags (B) and tube samples (QC), for both AO and PI stains

Figure 7: Adipocyte Diameter post-shipping comparison between bags (B) and tube samples (QC), for both AO and PI stain

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In this small sample study viability does not seem affected by delay to freezing, sample size, length of freezing, harvesting method or even shipping (Table 4).

Table 4: Postshipping large bag v. tube sample Adipocyte Viability(%) and Diameter (microns) Comparison

N R Min Max Mean Std Dev

Viability B (AO)

3

10.5

80.6

91.1

85.20

5.37

Viability QC (AO)

3 10.3 84.7 95.0 90.13 5.17

Viability B (PI)

3

13.1

68.9

82.0

76.87

7.00

Viability QC (PI)

3 35.0 60.10 95.10 76.36 17.63

Diameter B (AO)

3

7.10

27.15

34.25

30.13

3.68

Diamater QC (AO)

3 3.23 24.87 28.10 26.93 1.79

Diameter B (PI)

3

9.76

40-18

49.94

43.46

5.61

Diameter QC (PI)

3 4.92 35.31 40.23 38.02 2.50

B = large bag QC= Tube sample AO = Acridine Orange PI = Propidium Iodide

Viability seems to be well maintained throughout cryopreservation and thawing. The results in table 2 suggest that viability maybe even slightly increasing from pre-cryopreservation to post-thaw and post-shipping. This maybe explained by adipocyte mitosis. Indeed if cells were on the verge of dividing and are frozen in this

state, upon thawing, they can continue the process thus creating more cells and further proving the viability of the specimen. With regard to the significantly smaller diameter of the adipocytes post-thaw, this can be explained by the effect of the cryopreservant glycerol. Indeed glycerol as other cryopreservants tend to “dry out” the cell to preserve it. Too much water content can form crystals at those low temperatures, thus shearing the cell. Upon thaw, the glycerol is still in the cells for a while, which does not matter since the human body can tolerate glycerol well upon reim-plantation of the cells. Should that have been DMSO, cells would have had to be washed thoroughly prior to reimplantation, which would have been more problematic. CONCLUSION This pilot study demonstrated that the SOPs and processes of the accredited American Cryostem laboratory do safeguard adipocyte viability in a very satisfactory fashion. More studies are now underway to further explore the effect of the cryopreservation processes described on the byproducts of these fat samples, meaning SVF (Stromovascular Fraction), adipocyte stem cells and differentiated adipocytes, in light of the numerous therapeutic applications being developed as described above.

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