Ringer s lactate

Surgical preparation and blood gas measurements are detailed in Additional file 1.
The rats were randomized into four groups: (1) a sham operation group (control, n = 6), (2) a lipopolysaccharide (LPS) group (n = 7), (3) a LPS group with fluid resuscitation and a targeted mean arterial pressure (MAP) of 80–90 mmHg (LPS + FR, n = 7), and (4) a LPS group with blood transfusion and a targeted MAP of 80–90 mmHg (LPS + BT, n = 7). In the septic groups, a 30-min infusion of LPS (10 mg/kg; serotype 0127:B8; Sigma, The Netherlands) was given to induce septic shock. Fluid resuscitation was performed with a crystalloid solution (Ringer’s lactate; Baxter). Fluid resuscitation (median 4.64 ml, interquartile range 3–7 ml) and blood transfusion (median 4.19 ml, interquartile range 3–5.5 ml) (p = 0.2) were initiated 120 min after the end of LPS infusion and ended 15 min later. The experiment was ended 180 min after the end of LPS infusion or at the corresponding time point for the control group (Fig. 1). Of note, estimated blood volume in Wistar rats with a mean body weight of 325 g is 20.3 ml (from the equation of Lee and Blaufox [9 ]). Thus, 4.64 ml represents 22.8% of volemia and 4.19 ml represents 20.6% of volemia.Fig. 1

Experimental protocol. Time frame of the study. BT blood transfusion, FR fluid resuscitation, LPS lipopolysaccharide, MAP mean arterial pressure

In the SSCF facilities, AT was washed twice with Dulbecco’s phosphate-buffered saline with calcium and magnesium solution (DPBS +/+, Gibco, Life Technologies, Carlsbad, CA, USA) and enzymatically digested for 45 min at 37 °C under constant agitation in a cell culture incubator. For the latter, Liberase® MNP-S (Roche, Basel, Switzerland), i.e., a mix of collagenase I and II, and neutral proteases, was used. Enzymatic digestion was then stopped with sterile, clinical grade 1% human serum albumin (HSA, CSL Behring, King of Prussia, PA, USA) in DPBS without calcium and magnesium (DPBS −/−, Life Technologies); the hydrophilic phase was collected and subsequently filtered with 100-μm and 40-μm sieves. At the final step of SVF manufacturing, the hydrophilic phase was centrifuged and the resulting cell pellet, i.e., the autologous SVF, was resuspended in 5% HSA.
In the AP-HM facilities, the autologous SVF was obtained using the Celution 800/CRS automated processing system (Lorem Cytori, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, the collected lipoaspirate was washed with Ringer’s lactate (RL; Baxter, Deerfield, IL, USA) and enzymatically digested with Celase, a GMP cocktail of enzymes provided with consumables (Worthington Biochemical Corp., Lakewood, NJ, USA). The cells were concentrated, washed, aseptically recovered, and resuspended in RL.

Thiopentone sodium was obtained from Rhone Merieux (Harlow, Essex, UK). LPS (Escherichia coli, serotype 0127:B8) and all other compounds used in this study were purchased from Sigma-Aldrich Company Ltd (Poole, Dorset, UK), unless otherwise stated. All stock solutions were prepared using non-pyrogenic saline (0.9% [wt/vol] NaCl; Baxter Healthcare Ltd, Thetford, Norfolk, UK). Ringer’s lactate was also purchased from Baxter Healthcare. The bicinchoninic acid protein assay kit and SuperBlock blocking buffer were from Thermo Fisher Scientific Inc. (Rockford, IL, USA). Antibodies were from Cell Signalling Technology Inc. (Beverly, MA, USA).