Indication-based protocols with different solutions for mechanical stromal-cell transfer

Abstract

Background: Regenerative medicine is the fastest developing branch of plastic surgery in recent times. Adipose tissue is one of the largest and most important sources in the body for stromal cells. Although mechanical isolation methods are both very popular and have many advantages, they still have no accepted protocols.

Objective: We developed new protocols called indication-based protocols (IPs) for standardization and new techniques called mechanical stromal-cell transfer (MEST) by using ultra-sharp blades and dilution of adipose tissue with different solutions (saline, Ringer and 5% Dextrose)Methods & material: In order to obtain the desired physical structure (liquid, gel, solid) and the desired volume, four different types of IPs have been defined. Adipose tissue was prediluted with different solutions using 10 or 20 cc injectors in IPs 1 and 2, while condensed adipose tissue was used directly in IPs 3 and 4.

Results: In MEST, stromal cells were obtained from 100 mL of condensed fat using different IPs with 92% mean viability and cell counts of 26.80-91.90 × 10⁶. Stromal cells can be obtained in the desired form and number of cells by using four different IPs.

Conclusion: Isolation of stromal cells by cutting fat with sharp blades will prevent the death of fat tissue and stromal cells and will allow high viability and cell count with our new technique. Predilution with different solutions: Diluting the condensed adipose tissue with the desired solutions (saline, Ringer or 5% Dextrose) before the adinizing process will provide even more stromal cells.

Lay summary: Obtaining regenerative stromal cells from adipose tissue can be done by two methods: Enzymatic and mechanical. Mechanical methods have many advantages. Although mechanical stromal cell extraction from adipose tissue is very popular and many techniques have been described, there are still no accepted protocols, definition for the end product, and no consensus on the status of the stromal cells. In this study, stromal cells were obtained mechanically by using ultra-sharp blade systems, without exposing adipose tissue to blunt trauma. Thus, a higher number of cells and higher viability could be obtained. An "Indication based" protocol has been defined for the first time in order to obtain the desired number and status (solid, semi-solid, liquid)end product. Diluting the condensed adipose tissue with the desired solutions (saline, Ringer or 5% Dextrose) before the adinizing process will provide even more stromal cells. This will provide an opportunity for clinicians to obtain and apply a stromal cell solution for different indications in different anatomical regions.

Keywords: Stromal cells; adipose tissue; cell treatment; mechanical; stromal vascular fraction; total stromal-cells.

Figure 1.

(a) Adinizers: ultra-sharp blade system. Diameter of blades start from 4000 μ to 100 μ. (b) Schematic view of cross section of Adinizer two-sided blades. (c) View of blades of 100 μ Adinizer under microscope (×1000 magnification).

Figure 2.

Cell amount and viability results of IPs3 by LunaStem Automated Fluorescence Cell Counter device (Logos Biosystems, South Korea). Since TOST was solid form in IPs3, we have diluated 1/ 10 ratio. Nucleated cell amount should be accepted as 2.98 × 10⁶/mL.

Figure 3.

Cell amount and viability results of IPs3 by NAVIOS flow cytometer (Beckman Coulter, Miami, FL, USA).

Figure 4.

Final view of each IPs.

Figure 5.

Histopathological assessment of Adinized adipose tissue after adinized 100 μ blades and centrifugation in 1200 g, 6 min by Hematoxylin-Eosin staining. Cell integrity is still stable.

Figure 6.

(a) View of IPs1 after all process. There are four layers and stable adipose tissue above the TOST. (b) View of IPs3 after all process. There are 3 layers and there is stable adipose tissue above the TOST.