Vol.2, No.3, 80-91 (2013) Open Journal of Regenerativ e Medicine
http://dx.doi.org/10.4236/ojrm.2013.23012
Treatment of a mouse model of collagen
antibody-induced arthritis with human
adipose-derived secretions
Sinead P. Blaber1,2, Rebecca A. Webster1,2, Edmond J. Breen3, Graham Vesey2,
Benjamin R. Herbert1,2*
1Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, Australia;
*Corresponding Author:
2Regeneus Ltd., Gordon, Australia
3Australian Proteome Analysis Facility, Macquarie University, North Ryde, Australia
Received 2 August 2013; revised 2 September 2013; accepted 9 September 2013
Copyright © 2013 Sinead P. Blaber et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The use of adipose-derived cells as a treatment
for a variety of diseases is becoming increas-
ingly common. These therapies include the use
of cultured mesenchymal stem cells (MSCs) and
freshly isolated stromal vascular fraction (SVF)
alone, or in conjunction with other cells such as
adipocytes. There is a substantial amount of lite-
rature published on the therape utic properti es of
MSCs and their secretions as the main driver of
their therapeutic effect. However, there is little
data available on the therapeutic potential of
secretions from SVF, either with or without adi-
pocytes. We investigated the ability of secre-
tions from human adipose SVF alone and the
SVF co-cultured with adipocytes as a proxy for
cell therapy, to ameliorate an inflammatory dis-
order. This ethics approved study involved the
treatment of collagen antibody-induced arthritis
(CAIA) in mice with secretions from SVF, SVF
co-cultured with adipocytes, or a vehicle control
via both intravenous (IV) and intramuscular (IM)
routes. Treatment outcome was assessed by
paw volume, ankle size and clinical arthritis
score measurements. Serum samples were ob-
tained following euthanasia and analysed for a
p anel of 32 mouse cytokines and growth factors.
The dose and timing regime used for the IM ad-
ministration of both human secretion mixtures
did not significantly ameliorate arthritis in this
model. The IV administration of SVF adipocyte
co-culture secretions reduced the paw volume,
and significantly reduced the ankle size and
clinical arthritis score when compared to the IV
vehicle control mice. This was a superior thera-
peutic effect tha n treatment with SVF secretions.
Furthermore, treatment with SVF adipocyte co-
culture secretions resulted in a significant re-
duction in serum levels of key cytokines, IL-2
and VEGF, involved in the pathogenesis of rhe-
umatoid arthritis. Therefore, the SVF cocultured
with adipocytes is an attractive therapeutic for
inflammatory conditions.
Keyw ords: Collagen Antibody- I n d uced Arthritis
(CAIA); Stromal Vascular Fraction (SVF);
Adipocytes; Co-Culture; Secretions; Cytokines;
Growth Factors; Bio-Plex; Rheumatoid Arthritis
1. INTRODUCTION
The use of adipose-derived cells, including cultured
adipose-derived mesenchymal stem cells (MSCs), un-
cultured stromal vascular fraction (SVF) and SVF com-
bined with other cells such as adipocytes, is becoming
increasingly common for the treatment of a variety of
diseases. There are currently two distinct pathways, de-
fined by the regulatory environment, by which cell
therapies can proceed down to clinical use in a number
of major jurisdictions, including Australia, the UK and
some European countries. The first involves significant
safety and efficacy trials and is usually required for cell
therapies that involve allogeneic cells and in vitro expan-
sion or other forms of manipulation before administering
the cells to the patient. The second is a medical exemp-
tion regulatory category whereby a registered medical
practitioner can treat a patient in a clinic or hospital set-

S. P. Blaber et al. / Open Journal of Regenerative Medicine 2 (2013) 80-91 81
ting with a custom-made therapeutic for that patient pro-
viding the entire procedure is under the supervision of
the medical practitioner [1]. This alternative pathway
encompasses the use of autologous, freshly harvested tis-
sue that has only undergone minimal manipulation to
produce a cell population for therapeutic use in an in-
clinic procedure. The use of freshly isolated adipose-
derived cell populations such as the SVF, alone or in
combination with adipocytes, falls within this medical
exemption category. Consequently, the use of such cell
populations as autologous therapeutics is increasing.
Historically, the focus of cellular therapy has been de-
fining the ability of stem cells, including MSCs, to dif-
ferentiate into cells of the target issue type and their sub-
sequent potential therapeutic use. In recent years there
has been a shift in focus towards using fresh uncultured
cells for therapeutic use such as the SVF with or without
adipocytes partly due to the aforementioned regulatory
reasons. Furthermore, the realization that secretions are a
major driver of the therapeutic effect in mesenchymal
cellular therapy [2-4] has led to an increased focus on the
secretion capabilities of cells. Consequently the secretion
profiles produced by these isolated and mixed cell popu-
lations are of interest in order to delineate functional dif-
ferences and guide potential therapeutic use of cellular
therapies. These adipose-derived secretions may also be
of importance for future off-the-shelf secretion based
therapeutics for use when cellular therapy may not be
possible or appropriate, or in some cases may be used in
conjunction with cellular therapy.
Adipose tissue is comprised of a variety of different
cell types, which reside in close proximity to each other
in vivo. This allows for substantial cross-talk between
these different cell types though cell-cell contact, surface
molecular receptors and secreted factors. This commu-
nication between the cells is essential for the normal
functioning of adipose tissue and this complex-reciprocal
signaling has been demonstrated by in vitro co-culture
experiments [5-7]. We have previously demonstrated that
unique secretion profiles can be obtained from different
adipose-derived cell populations [8]. In particular, we
demonstrated that the co-culture of adipocytes with the
SVF results in the cells acting in a synergistic manner
and consequently increasing the production of 9 cyto-
kines when compared to culturing the SVF alone [8]. For
example, IL-1Ra was produced in significantly increased
levels by the co-culture of SVF with adipocytes versus
the SVF alone. IL-1Ra is one of the most important
naturally occurring anti-inflammatory cytokines because
it blocks the actions of the potent pro-inflammatory cy-
tokine, IL-1β. Consequently, IL-1Ra is currently being
trialed as a treatment for diseases with a significant in-
flammatory component, such as autoimmune disorders.
The administration of a single cytokine or growth fac-
tor has been trialed in animals with induced diseases and
humans with clinical conditions. This treatment approach
has frequently resulted in the development of adverse
reactions [9-11]. For example, the administration of IL-
12 to cancer patients in a Phase II clinical trial caused
serious side effects including renal, neurological, hepatic
hematopoietic and cardiac toxicities resulting in the hos-
pitalization of 12 patients and the death of 2 patients [9].
In this case, the administration of IL-12 caused an inap-
propriate immune response and a profound up-regulation
of IFN-γ [9]. In contrast, some biologics, in particular the
administration of recombinant IL-1Ra, have not demon-
strated any efficacy, or only a modest effect, for auto-
immune disorders [see 12 and references therein]. To this
end, studies have demonstrated that treatment with two
bioactive factors results in a superior therapeutic out-
come than the administration of these factors individu-
ally [13,14]. There are numerous in vitro studies illus-
trating the immuno-modulatory, angiogenic, mitogenic,
anti-apoptotic, and anti-scarring properties of both adi-
pose-derived and bone marrow-derived MSCs [see 15
and references therein]. Furthermore, administration of
the secretions from these MSCs has been shown to im-
prove corneal wound healing following chemical burns
[16], and recovery from myocardial infarctions [17] and
ischemic brain injuries [18]. These studies demonstrate
the therapeutic potential of MSC secretions for the treat-
ment of non-autoimmune conditions, however there is
little data available on the ability of secretions to ame-
liorate autoimmune conditions. Additionally, there is
limited data available on the treatment of conditions in
vivo with cells or secretions from mixed cell populations
such as the SVF, alone or in combination with adipo-
cytes. As we have previously demonstrated that the in
vitro co-culturing of adipocytes with the SVF results in a
distinct secretion profile when compared to culturing the
SVF alone [8], the in vivo comparison of this result is of
interest. Given the promising data available on condi-
tioned media for injury models, we were interested in its
efficacy for autoimmune conditions. Our interest was to
understand the efficacy of human adipose derived cells
for inflammatory autoimmune conditions. Cultured
MSCs do not usually elicit an immune response, even
when used as a xeno-transplant. This would not be the
case, however, for a mixed population of human cells in
immuno-competent mice. In order to test the likely
therapeutic effect of human SVF with and without adi-
pocytes, we chose to use conditioned media alone, thus
avoiding any immunological issues with human cells.
Collagen antibody-induced arthritis (CAIA) in mice is
an inflammatory disorder that shares many pathological
and histological similarities with clinical rheumatoid
arthritis in humans, including synovitis with infiltration
of polymorphonuclear and mononuclear cells, pannus

S. P. Blaber et al. / Open Journal of Regenerative Medicine 2 (2013) 80-91
82
formation, fibrosis cartilage degradation and bone ero-
sion [19]. Furthermore, significant swelling and redness
is observed in the paws of mice suffering from CAIA
[20]. CAIA is induced by the administration of a cocktail
of 5 anti-type II collagen antibodies, followed by a
lipopolysaccharide (LPS) injection three days later. The
subsequent administration of LPS following the antibody
cocktail not only increases the severity of the arthritis
through the induction of pro-inflammatory cytokines and
complement component activation, but also reduces the
amount of monoclonal antibody required to induce the
arthritis in this model [20 and references therein, 21].
Arthritis in the CAIA model development is rapid and
relatively synchronized through the synchronization of
antibody administration [19].
The primary objective of this work was to perform an
in vivo study to investigate the ability of human adi-
pose-derived secretions, as a proxy for human SVF cell
therapy, to ameliorate an inflammatory disorder. The
SVF cell secretions, with or without adipocyte co-culture,
are a complex mixture of proteins and other signaling
factors. Many of the cytokines secreted by adipose SVF
have pleiotropic effects, which combined with the large
number of cytokines would make any attempt at eluci-
dating the mechanism of action very difficult. The goal
of this study was to investigate the likely therapeutic
benefit of SVF cellular therapy rather than an attempt to
identify key cytokines. As there is limited in vivo data
available on the use of secretions from mixed cell popu-
lations such as adipose SVF, we chose to pursue a pre-
liminary study to compare the ability of human SVF
co-cultured with adipocytes (referred to as SVF adipo-
cyte co-culture) and SVF secretions to ameliorate arthri-
tis in a CAIA model in mice. Furthermore, we investi-
gated whether administration via the intravenous (IV) or
intramuscular (IM) route had any effect on the ability of
these adipose-derived secretions to reduce the severity of
arthritis in this mouse model.
2. MATERIALS AND METHODS
2.1. Production of Secretions from Human
Lipoaspirate
This research, involving obtaining human adipose tis-
sue, and administering the resultant human secretions to
animals, was approved by the Macquarie University hu-
man research ethics committee. Written consent was
obtained from the patient who provided their lipoaspirate
sample after undergoing a routine liposuction procedure
for cosmetic reasons. The lipoaspirate sample was proc-
essed as previously described [8]. Briefly, 200 g of li-
poaspirate was digested with 0.5 mg/mL collagenase
(Lomb Scientific, USA) in saline in a 37˚C water bath
for 30 min with periodic mixing. The digested samples
were passed through an 800 μm mesh and centrifuged at
1500 × g for 5 min to obtain the pelleted cells (SVF) and
floating adipocytes. The floating free lipid layer was as-
pirated and discarded. The adipocyte and SVF fractions
were washed separately with saline and centrifuged at
1500 × g for 5 min. The SVF pellet was resuspended in
our Standard Media that consisted of high glucose Dul-
becco’s Modified Eagle Medium (HG DMEM; Invi-
trogen, USA) supplemented with 10% foetal bovine se-
rum (FBS; Bovogen, Australia) and 1% Penicillin-
Streptomycin solution (Invitrogen, USA). A portion of
the SVF pellet was filtered through a 35 μm nylon mesh
topped tube (Becton Dickinson, USA). SVF samples
were enumerated and the viability determined in Tru-
Count tubes (Becton Dickinson, USA) containing iso-
flow (Becman Coulter, USA) propidium iodide (10
μg/mL; Sigma, USA) and Syto11 (1 μM; Invitrogen,
USA) using a FacsScan flow cytometer (Becton Dickin-
son, USA). The total number of viable cells in the SVF
pellet was determined.
Two T175 cm2 culture flasks were each seeded with
29 million viable SVF cells. The SVF adipocyte co-cul-
ture flask also received 30 mL of adipocytes. As the
adipocytes transferred to the SVF adipocyte co-culture
flask were floating saline, the SVF flask received 30 mL
of saline. The volume in each T175 cm2 flask was nor-
malized to a total of 80 mL with Standard Media. The
flasks were incubated at 37˚C with 5% CO2 for 72 hours
and the conditioned medium collected, centrifuged at
4980x g for 10 min and stored at −80˚C. These condi-
tioned medium samples were thawed, filter sterilized
using 0.22 µm syringe filters, aliquoted and frozen at
−80˚C. These aliquoted samples were administered to
mice suffering from CAIA.
2.2. Collagen Antibody-Induced Arthritis—
Mouse Model
A commercial company, TetraQ, based at the Univer-
sity of Queensland, conducted this CAIA trial. The Uni-
versity of Queensland’s animal ethics committee ap-
proved this trial involving the administration of human
adipose-derived secretions to mice suffering from CAIA.
The trial was blinded and was performed at the TetraQ
premises in accordance with the guidelines set out in the
Australian Code of Practice for the Care and Use of
Animals for Scientific Purposes, 7th edition, 2004. Nul-
liparous and non-pregnant female Balb/c (BALB/
cJAsmu) mice at 6 - 8 weeks of age were used in this
trial. The mice had access to rat and mouse pellets (Spe-
cialty feeds, Australia), chewing sticks (Able Scientific,
Australia), Alphatwists (Tecniplast, Australia) and water
ad libitum throughout the study. The housing tempera-
ture throughout the study was 23˚C ± 3˚C, with a humid-
ity of 30% - 70%, a 12-hour light/dark cycle and a

S. P. Blaber et al. / Open Journal of Regenerative Medicine 2 (2013) 80-91 83
minimum of 15 air changes per hour. The mice were
allowed to acclimatize to these housing conditions for 3
days prior to commencement of the trial.
At day zero, each mouse (total of 36 mice) received an
intravenous injection of 1.5 mg (150 μL) of an anti-type
II collagen 5 clone antibody cocktail (Chondrex Inc.,
Australia). On day 3, mice received an intraperitoneal
injection of 80 μL (40 μg/mouse) of LPS. The trial de-
sign consisted of 6 groups, each containing 6 mice (Ta-
ble 1). Groups 1, 2, and 3 received 100 µL of human
SVF adipocyte co-culture secretions, human SVF secre-
tions or the vehicle control (containing HG DMEM and
1% P/S) respectively via the intravenous (IV) route on
days 6, 7 and 8. Groups 4, 5 and 6 received 50 µL of
human SVF adipocyte co-culture secretions, human SVF
secretions or vehicle control respectively via the intra-
muscular (IM) route every second day for 4 days (days 6,
8, 10 and 12).
The mice were monitored throughout the trial period
(2 weeks) and paw volume, ankle size and clinical arthri-
tis score measurements were taken on days 0 and 2 - 13.
The measurements were taken prior to administration of
the collagen antibody cocktail (days 0) and LPS (day 3)
and prior to administration of vehicle and the test articles
(human SVF adipocyte co-culture secretions and SVF
secretions). The paw volume of each mouse was meas-
ured using a plethysmometer. Plethysmometers are rou-
tinely used to measure small volume changes in rodent
paws by measuring the water displacement following
immersion of the paw in a specialized chamber which is
attached to a digital monitor [22]. The paw size was
measured using microcalipers across the hillock (ankle
joint) of each hindpaw. The mice were assessed for the
severity of arthritis using a standard scale [23] (0—nor-
mal; 1—mild redness, slight swelling of ankle or wrist,
redness and swelling limited to individual joints; 2—
moderate swelling of ankle or wrist, redness in more than
one joint, 3—severe swelling including some digits, an-
kle or foot; 4—maximal swelling and inflamed, involve-
ing multiple joints). Following euthanasia with Letha-
barb at day 14, blood was collected from all mice via-
cardiac puncture. Blood was allowed to clot at room
temperature for 30 min prior to centrifuging at 2500 × g
for 10 min to obtain serum. Serum samples from age-
matched naïve mice were also obtained as control sam-
ples. Serum samples were stored at −80˚C prior to analy-
sis using Bio-Plex technology.
2.3. Bio-Plex Analysis of Serum Samples
A total of 32 mouse cytokines were measured in the
serum samples of mice treated IV with the vehicle con-
trol or human secretion mixtures to investigate the effect
of the various treatment regimes on the mice. The SVF
and SVF adipocyte co-culture secretion mixtures, vehicle
control, media control and all IV treated mouse serum
samples (including the naïve mouse serum samples) were
filtered through 0.20 m Nanosep MF Centrifugal De-
vices with Bio-Inert® Membrane (Pall Scientific, USA)
for 5 min at 9000 x g. Samples (50 μL) of each filtered
sample were analysed using the Bio-Plex Pro Mouse
Cytokine 23-plex and Bio-Plex Pro Mouse Cytokine
9-plex assays (Bio-Rad, USA) according to the manu-
facturers instructions. The washing steps were performed
on the Bio-Plex Pro II magnetic wash station and the
data was acquired using the Bio-Plex 200 system with
version 5.0 software (Bio-Rad, USA). Mouse IL-1Ra
cross-reacts with the human IL-1Ra antibody in the Bio-
Plex Pro Human Cytokine 27-plex assay. Therefore IL-
1Ra was measured in the mouse serum samples using
this human kit and the same methodology described
above.
2.4. Data Anal ysis
2.4.1. Dat a Analysis of CAIA Primary Outcome
Measurements
The primary outcome measures, paw volume (cm3),
ankle size (mm), and clinical arthritis score in each group
are presented as the mean ± standard error of the mean
(SEM). The Delta (Δ) clinical arthritis score, paw vol-
ume and paw size was calculated by subtracting the pre-
treatment score from the post-treatment scores. Negative
Table 1. Trial design of treatment groups of mice suffering from collagen antibody-induced arthritis.
Group (6 mice/group) Treatment Route of administration Volume administered Treatment regime
Group 1 SVF adipocyte co-culture secretions
Group 2 SVF secretions
Group 3 Vehicle
Intravenous (IV) 100 µL Days 6, 7 and 8
Group 4 SVF adipocyte co-culture secretions
Group 5 SVF secretions
Group 6 Vehicle
Intramuscular (IM) 50 µL Days 6, 8, 10 and 12

S. P. Blaber et al. / Open Journal of Regenerative Medicine 2 (2013) 80-91
84
values were given a value of 0. The Area Under the Δ
clinical arthritis score, paw volume and paw size versus
day curves were determined for each group as a measure
of the extent of action and duration of arthritis. The
non-parametric Dunnett’s Multiple Comparison test was
performed on Δ clinical arthritis score, paw volumeand
paw size area under the curve (AUC) values for groups
of mice administered either SVF adipocyte coculture
secretions or SVF secretions relative to the correspond-
ing values for animals administered vehicle controls. The
Graphpad Prism data analysis software package (version
5.03) was used for all data and statistical analysis. The
statistical significance criterion was P < 0.05. A Welch
two-tailed t-test was performed on the clinical score raw
data between mice in the IV SVF adipocyte co-culture
and SVF secretion treatment groups.
2.4.2. Data Analysis of Bio-Plex Datasets
The human SVF adipocyte co-culture and SVF secre-
tion mixtures were run on both mouse Bio-Plex kits as
controls. We observed some cross-reactivity, however,
this should have no impact on the study because the cir-
culating half-life of cytokines is usually minutes to hours,
although it differs for individual cytokines [24]. In this
study, the serum was collected from mice 6 days after the
last IV administration of human secretions. Therefore,
we have assumed 0% of the human cytokines adminis-
tered to the mice remained in their circulation at the
point of euthanasia.
The mean ± SEM values of all cytokines measured
were determined for each of the groups (n = 6 for all IV
treatment groups, n = 3 for naïve mice). A two-tailed
t-test was used to test the significance between the aver-
age concentrations of each cytokine in the IV treatment
groups (SVF adipocyte co-culture secretions and SVF
secretions) to the vehicle control group. p-values < 0.05,
denoted with a*, were considered statistically significant
compared to the corresponding vehicle control group.
Additionally, a two-tailed t-test was also used to test the
significance between the average concentrations for each
cytokine in the treatment groups (SVF adipocyte co-
culture secretions and SVF secretions) and the vehicle
control group, when compared to the naïve mice serum.
These statistically significant results with a p-value <
0.05 are denoted with a^.
3. RESULTS
3.1. Primary Outcome measures
3.1.1. IM
Figure 1 illustrates that administration of SVF adipo-
cyte co-culture and SVF secretions IM slightly reduced
the primary outcome measures of paw volume (Figure
1(a)), ankle size (Figure 1(d)) and clinical arthritis score
(Figure 1(g)) when compared to the mice that received
the vehicle control IM. However, an analysis of the Δ
area under the curve (ΔAUC) results for these primary
outcome measures (Figures 1(c), (f) and (i) over the
course of the trial demonstrates that the reduction in
these measurements following treatment with SVF adi-
pocyte co-culture or SVF secretions was not statistically
sig- nificant. Whilst these results were promising, a bet-
ter clinical outcome may be obtained after optimization
of the dose and timing of the IM treatment regime with
SVF adipocyte co-culture or SVF secretions.
3.1.2. IV
Figure 1 contains the results of the primary outcome
measures from the mice treated with the vehicle control,
SVF adipocyte co-culture secretions or SVF secretions
via the IV route. From these graphs it is evident that ad-
ministration of SVF adipocyte co-culture and SVF secre-
tions reduced the paw volume (Figure 1(b)), ankle size
(Figure 1(e)) and clinical arthritis scores (Figure 1(f)) of
mice suffering from CAIA when compared to the vehicle
control group. In particular, the average clinical arthritis
score of the mice treated with SVF adipocyte co-culture
secretions was the only treatment group to achieve a re-
duction in clinical arthritis score to baseline following
the establishment of severe arthritis (Figure 1(h)). The
analysis of the ΔAUC results demonstrated that the av-
erage paw volume throughout the course of the study
was reduced in the mice treated IV with SVF adipocyte
co-culture secretions when compared to the IV vehicle
control although this reduction was not statistically sig-
nificant (Figure 1(c)). The ΔAUC ankle size was sig-
nificantly reduced in both the SVF adipocyte co-culture
and SVF secretion treatment groups when compared to
the IV vehicle control (Figure 1(f)). Looking at an over-
all measure of effect, the IV administration of the SVF
adipocyte co-culture secretions resulted in a significant
reduction in the ΔAUC clinical arthritis score (Figure
1(i)). Furthermore, the SVF adipocyte co-culture secre-
tions significantly (p-value = 0.005 using a Welch two-
sided t-test) reduced the clinical arthritis scores of mice
when compared to the SVF secretion treatment group.
These results demonstrate that the IV administration of
secretions derived from adipose cell populations resulted
in the reduction of all measurements of arthritis in CAIA
mice when compared to the vehicle control mice. Spe-
cifically, IV treatment with secretions from the SVF
co-cultured with adipocytes produced the largest effect,
with a reduction in the paw volume and a significant
reduction in the ankle size and clinical arthritis score
when compared to the vehicle control group.
3.2. Serum Levels of Mouse Cytokines and
Growth Factors
For these experiments the serum from naïve mice was

S. P. Blaber et al. / Open Journal of Regenerative Medicine 2 (2013) 80-91 85
used as an indicator of normal physiological cytokine
levels. Figure 2 contains the serum levels of the pro- and
anti-inflammatory cytokines measured in the naïve mice,
and the mice treated IV with the vehicle control, SVF
adipocyte co-culture or SVF secretions. IL-1α was pre-
sent in significantly higher levels in the serum of the
mice treated with SVF adipocyte co-culture secretions
when compared to the IV vehicle control group (Figure
2(a)). Additionally the levels of IL-1α in the mice treated
with SVF adipocyte co-culture and SVF secretions were
significantly higher than the levels measured in the naïve
mice (Figure 2(a)). The concentration of IL-1β measured
in the vehicle control and both secretion treatment
groups were significantly higher than the physiological
levels seen in the naïve mice (Figure 2( b)). The level of
IL-5 measured in the vehicle control group was signifi-
cantly higher than the level measured in the naïve mice
(Figure 2(c)). In comparison to the vehicle control mice,
the serum of mice treated with SVF adipocyte co-culture
and SVF secretions contained significantly reduced lev-
els of IL-5 (Figure 2(c)). TNF-α was measured in sig-
nificantly higher levels in the vehicle control and SVF
adipocyte co-culture groups when compared to the naïve
mice (Figure 2(d)). Whilst treatment with SVF and SVF
adipocyte co-culture secretions increased the levels of
the anti-inflammatory cytokine, IL-1Ra, this increase
was not statistically significant (Figure 2(e)). The levels
of IL-10 and IL-13 were significantly increased in the
mice treated with SVF adipocyte co-culture secretions
when compared naïve mice (Figures 2(f) and (g) respec-
tively). IL-2 and IL-6 are considered to have pro-in-
flammatory or anti-inflammatory roles under different
circumstances. Both IL-2 and IL-6 were present in sig-
nificantly increased concentrations in the serum of the
vehicle control group mice when compared to the naïve
mice (Figures 2(h) and (i) respectively). However, sig-
(a) (b) (c)
(d) (e) (f)
(g) (i)
(h)
Figure 1. Primary outcome measures of mice suffering from collagen antibody-induced arthritis treated with secretions from SVF
co-cultured with adipocytes, SVF secretions or vehicle controls. Mice were induced with collagen antibody-induced arthritis and
subsequently treated with a vehicle control, SVF adipocyte co-culture secretions or SVF secretions via the intramuscular (IM) or
intravenous (IV) route of administration. Paw volume (a)-(b), ankle size (d)-(e) and clinical arthritis score (g)-(h) were measured in
the mice throughout the course of the trial. These results are expressed as the absolute mean ± standard error of the mean (SEM; n =
6). The mean ± SEM Δ area under the curve (ΔAUC) trial results for the paw volume (c), ankle size (f), and clinical arthritis score (i)
were calculated for each treatment group. The ΔAUC results obtained from each treatment group were compared to the correspond-
ing vehicle control administered by the same route. a* denotes a p-value of <0.05 when compared to the IV vehicle control group.

S. P. Blaber et al. / Open Journal of Regenerative Medicine 2 (2013) 80-91
86
(a) (b) (c)
(d) (e) (f)
(g) (h) (i)
Figure 2. Serum levels of pro- and anti-inflammatory cytokines measured in mice suffering from collagen antibody-induced arthritis
following IV treatment with secretions from the SVF co-cultured with adipocytes, SVF secretions or the vehicle control. Serum sam-
ples were collected following euthanasia of the mice in all IV treatment groups at the completion of the trial and from naïve mice.
The results of the pro-inflammatory cytokines, IL-1α (a), IL-1β (b), IL-5 (c) and TNF-α (d), the anti-inflammatory cytokines IL-1Ra
(e), IL-10 (f) and IL-13 (g), and the cytokines with dual roles IL-2 (h) and IL-6 (i) in each control or treatment group are graphed as
the mean ± standard error of the mean (SEM; n = 6 for treatment groups; n = 3 for naïve serum samples). a* or ^ denotes a p-value of
< 0.05 when compared to the vehicle control or naïve groups respectively.
nificantly reduced concentrations of IL-2 were measured
in the SVF adipocyte co-culture secretion treatment
group when compared to the vehicle control group (Fig-
ure 2( h)). The chemokine eotaxin was measured in sig-
nificantly increased concentrations in the mice treated
with secretions from the SVF co-cultured with adipo-
cytes and the SVF alone when compared to both the ve-
hicle control and naïve groups (Figure 3(a)). KC, MIP-
1β and GM-CSF were measured in significantly ncreased
levels in the mice treated with SVF secretions versus the
naïve mice (Figures 3(b), (e) and (f) respectively).
MCP-1 and PDGF-bb levels were significantly increased
in the serum of the mice treated with SVF adipocyte
co-culture secretions when compared to the naïve mouse
group (Figures 3(c) and (g) respectively). MIG was pre-
sent in significantly reduced concentrations in the SVF
adipocyte co-culture treatment group when compared to
the vehicle control group (Figure 3(d)). VEGF was
measured in significantly increased levels in the vehicle
control group when compared to the naïve mice group
(Figure 3(h)). Treatment with SVF adipocyte co-culture
secretions significantly reduced the serum levels of
VEGF of the mice when compared to the vehicle control
(Figure 3(h)).
4. DISCUSSION
4.1. SVF Adipocyte Co-Culture Secretions:
A Promising Therapeutic for
Inflammatory Conditions
Rheumatoid arthritis is an autoimmune disorder char-
acterized by chronic inflammation involving a shift to-
wards the Th1 inflammatory response. The components
of the Th1 response are involved in initiating and main-
taining inflammation, including the production of the
pro-inflammatory cytokines, IFN-γ, IL-1α, IL-1β, IL-2,

S. P. Blaber et al. / Open Journal of Regenerative Medicine 2 (2013) 80-91 87
(a) (b)
(c) (d)
(e) (f)
(g) (h)
Figure 3. Serum levels of chemokines and growth factors
measured in mice suffering from collagen antibody-induced
arthritis following IV treatment with secretions from the SVF
co-cultured with adipocytes, SVF secretions or a vehicle con-
trol. Serum samples were collected following euthanasia of the
mice in all IV treatment groups at the completion of the trial
and from naïve mice. The results of the chemokines, Eotaxin
(a), KC (b), MCP-1 (c), MIG (d), and MIP-1β (e), and the
growth factors GM-CSF (f), PDGF-bb (g) and VEGF (h), in
each control or treatment group are graphed as the mean ±
standard error of the mean (SEM; n = 6 for treatment groups; n
= 3 for naïve serum samples). a* or ^ denotes a p-value of <0.05
(students t-test) when compared to the vehicle control or naïve
groups respectively.
IL-12 and TNF-α [25,26]. These cytokines have been
implicated in the pathogenesis of rheumatoid arthritis
and are considered to contribute to cartilage degradation
and bone erosion in this disease [27-29]. Routinely used
glucocorticoid therapy promotes a shift towards the Th2
response [30], thereby counterbalancing the Th1 skewed
environment seen in rheumatoid arthritis patients [31].
MSC therapy has been shown to have a positive effect
on autoimmune conditions, primarily via secreted factors
and their ability to induce peripheral tolerance [32,33].
We have previously demonstrated that the in vitro co-
culture of SVF with adipocytes results in the production
of a distinct secretion profile involving significantly in-
creased concentrations of IL-1Ra, IL-6, IL-7, IL-12,
IFN-γ, IP-10, bFGF, G-CSF and VEGF when compared
to culturing the SVF alone [8]. The in vitro effect of
co-culture was carried over into the animal model used in
this study and the IV administration of SVF adipocyte
co-culture secretions achieved a superior therapeutic
outcome. In this study the secretions administered were a
complex mixture of hundreds of proteins and other sig-
naling molecules, so the precise mechanism of action of
this treatment is not known. A reduction of the clinical
score to baseline at day 12 was observed in the CAIA
mice treated with the SVF adipocyte co-culture treatment
group. If it is assumed the circulating half-life of the hu-
man cytokines administered to the CAIA mice was min-
utes to hours [24], this reduction of the clinical score
occurred 6 days after the human cytokines were cleared
from the circulation. One of the mechanisms of action of
MSCs is their ability to induce peripheral T cell tolerance
by suppressing T cell proliferation and inducing T regu-
latory cell production [32,34]. The ability to induce T
cell tolerance is an important therapeutic strategy for the
effective treatment of autoimmune disorders. As secre-
tions from MSCs are the major driver of the angiogenic,
anti-apoptotic and anti-scarring properties of these cells,
it is likely that MSC-derived cytokines also play some
role in their ability to induce T cell tolerance. There is
evidence that MSCs secrete cytokines in an environment
or injury specific manner, with TGF-
 and IL-10 being
important for inducing tolerance in autoimmune condi-
tions such as RA [35,36]. As discussed by Ichim et al.,
(2010) the SVF contains a variety of cell types including
T regulatory cells and high levels of MSCs [34]. There-
fore, in this study, the SVF adipocyte co-culture and SVF
secretion mixtures are likely to contain cytokines pro-
duced from both MSCs and T regulatory cells that are
involved in inducing T cell tolerance. However, the
therapeutics used in this study were complex mixtures of
cytokines, which cannot respond to the local environ-
ment in the same dynamic way as implanted cells. It is
possible, therefore, that the balance of cytokines admin-
istered induced only partial tolerance in these mice and
that repeated doses would be required if symptoms re-
curred. This may explain the improvement in the thera-
peutic effect observed 4 days after the administration of
the SVF adipocyte co-culture secretions. If SVF cell
therapy was used as the therapeutic it is anticipated that
the MSCs would induce T cell tolerance over a longer
post-treatment period [34,37] Although it is likely that
the effects observed with conditioned media will be
similar to SVF cell therapy, there will be some differ-
ences. After the infusion of a mixed population of cells,
such as SVF with adipocytes the subsequent therapeutic
effect appears to be separated into two related phases.
Firstly, the mixed population of cells secretes im-

S. P. Blaber et al. / Open Journal of Regenerative Medicine 2 (2013) 80-91
88
muno-modulatory cytokines, which have important the-
rapeutic roles in reducing inflammation and pain. Sec-
ondly, the high levels of MSCs in the SVF are likely to
embed in the host tissue, and control the microenviron-
ment through their paracrine activities. It is anticipated
that the therapeutic effect will be similar, but longer last-
ing, when human adipose-derived cell populations, such
as SVF and adipocytes are used.
As observed in Figure 1(h) the clinical scores are in-
creasing at the last time-point, which may indicate a re-
currence of symptoms in some mice. In addition to the
direct effect of cytokines on peripheral tolerance, it is
also possible that the significantly higher levels of G-
CSF in the SVF adipocyte co-culture secretions over the
SVF secretions administered to the CAIA mice may play
some role. G-CSF has classically been used to stimulate
mobilization of hematopoietic stem cells from the bone
marrow into the circulation. More recently, mobilization
of MSCs from the bone marrow has been demonstrated
following administration of G-CSF [38]. Therefore, it is
possible that following administration of the therapeutics
used in this study, the high levels of G-CSF stimulated
mobilization of some MSCs from the bone marrow,
which may have contributed to the therapeutic outcome
observed.
Our results suggest that the treatment with secretions
from adipose-derived populations may be modulating the
immune response and reducing the inflammation in these
mice. At the serum level, it appears that the therapeutics
used did not promote an exclusive Th1 or Th2 response.
Instead, a significant increase in the pro-inflammatory
cytokines IL-1α, IL-1β and TNF-α and a corresponding
increase in the anti-inflammatory cytokines, IL-1Ra,
IL-10 (p < 0.05) and IL-13 (p < 0.05) were observed in
the SVF adipocyte co-culture treatment group.
The role of IL-5 in rheumatoid arthritis is not well un-
derstood. However, whilst serum levels of IL-5 are gen-
erally < 10 pg/mL, a significant increase is seen between
early stage rheumatoid arthritis patients and healthy peo-
ple [39]. Although the mouse serum levels of IL-5 in this
study were higher than the levels seen in people, IL-5
was significantly increased in the vehicle control group
whereas the mice treated with the therapeutics had ap-
proximately physiological levels. Rheumatoid arthritis
patients have increased serum levels of the chemokine
eotaxin [39], and higher levels been associated with less
radiographic progression [40]. In this mouse model of
rheumatoid arthritis, the mice in the vehicle control
group that developed the most severe arthritis did not
have elevated serum eotaxin levels above physiological
levels. However, eotaxin levels were significantly in-
creased in the mice that received the therapeutics in this
study when compared to both the naïve and vehicle con-
trol mice (Figure 3(a)). Therefore whilst there are as-
pects of the eotaxin and IL-5 results that are consistent
with rheumatoid arthritis patients, the exact role of these
two cytokines in CAIA is not well understood.
IL-2 and VEGF have been implicated in the patho-
genesis of rheumatoid arthritis patients and are signifi-
cantly increased in the serum of human patients with
established rheumatoid arthritis [39]. Consistent with this
observation, the serum levels of IL-2 and VEGF were
significantly increased in the vehicle control group of
mice that developed severe arthritis, when compared to
physiological levels. IL-2 is a Th1 derived cytokine that
contributes to disease progression by exacerbating Th1-
mediated disease states whilst recruiting other immune
cells to the joint [41,42]. Increased expression of the IL-2
receptor on the surface of T cells is seen in a number of
autoimmune disorders including rheumatoid arthritis [43].
It is known that blocking the ability of IL-2 to bind to its
receptor does not affect natural immunity but can induce
immunosuppression in a broad range of T cell mediated
diseases [44]. Consequently, the IL-2 receptor is being
targeted as a therapeutic strategy for the treatment of
autoimmune disorders [43,44]. The administration of
SVF adipocyte co-culture secretions produced an anti-
inflammatory effect and we observed significantly lower
serum levels of IL-2 in this treatment group, consistent
with the reduced severity of arthritis in these mice.
Although the etiology of rheumatoid arthritis disease
development is not known, one of the characteristic fea-
tures of early rheumatoid arthritis is an increase in the
number of blood vessels in the pannus developing from
the synovium [45]. The increased density of blood ves-
sels is thought to result from a dis-regulation and imbal-
ance between pro- and anti-angiogenic stimuli [45]. To
this end, significantly increased circulating levels of
VEGF are seen in patients with established rheumatoid
arthritis when compared to healthy controls [39]. Con-
sequently, blocking angiogenesis and the pathogenic
mechanisms that stimulate neovascularization is an at-
tractive therapeutic strategy and one that is being trialed.
In fact, two studies have demonstrated that administra-
tion of the VEGF soluble receptor to mice suffering from
collagen-induced arthritis reduces the severity of the
disease [46,47]. In this study, the SVF adipocyte co-cul-
ture secretions contained significantly higher concentra-
tions of VEGF than the levels present in the SVF secre-
tions. However, we observed the largest improvement
over the course of the study in the group of mice that
received the SVF adipocyte co-culture secretions and this
was accompanied by a significant reduction in serum
levels of VEGF when compared to the vehicle control
mice. Clearly the negative consequences resulting from
angiogenesis require more than short-term elevated
VEGF levels. The half-life of VEGF is approximately 34
minutes [48] and angiogenesis typically requires many

S. P. Blaber et al. / Open Journal of Regenerative Medicine 2 (2013) 80-91 89
days or weeks [49].
4.2. Secretions-Based Therapy: Therapeutic
Importance
The use of autologous cell therapy, particularly with
minimally manipulated adipose cell populations is be-
coming increasingly common as a therapy option for a
variety of diseases including inflammatory conditions.
The secretion of immuno-modulatory and trophic factors
are a key aspect of the in vivo effect of MSCs and mixed
cell populations [15]. We have previously demonstrated
in vitro that mixed populations from adipose tissue, par-
ticularly those with SVF and adipocytes, produce distinct
secretion profiles with high levels of growth factors and
anti-inflammatory cytokines. The in vivo assessment of
adipose SVF adipocyte co-culture secretions as not only
a proxy for human SVF cell therapy, but also a potential
stand-alone therapeutic in the CAIA model, enabled us to
confirm our in vitro data. Although the exact mechanism
of the therapeutic effect of the administration of SVF
adipocyte co-culture secretions to CAIA mice is not
known, no adverse effects were seen throughout the trial.
5. CONCLUSION
The administration of secretions from adipose-derived
cell populations as a proxy for human adipose SVF cell
therapy, do have the ability to reduce the severity of in-
flammatory diseases, although the mixture of cytokines
is likely to be important. MSC therapy has been shown to
be safe [50] and under the medical exemption regulatory
category, autologous cell therapies, such as freshly iso-
lated adipose-derived SVF with adipocytes, can be per-
formed in-clinic under the supervision of a registered
medical practitioner [1]. It is anticipated that the thera-
peutic effect will be similar, but longer lasting, when
human adipose-derived cell populations, such as SVF
and adipocytes are used. Additionally, the administration
of these secretions to CAIA mice demonstrated a benefi-
cial therapeutic effect thereby also providing a basis for
future off-the-shelf secretion based products. There are a
number of advantages to using off-the-shelf secretion
based products as a therapeutic which includes the ease
of transporting and storing these products and the ease of
delivery via either the IV, IM, or sub-cutaneous route.
However, due to the relatively short half-life of cytokines,
it is likely that multiple treatments with secretion-based
products will be required to sustain the therapeutic effect.
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