Sample PDF Manuscript for Protocol Submission

Manuscript Title: Isolation and Culture of Human Adipose-Derived Mesenchymal Stem Cells (AD-MSCs)

Author(s): Jane A. Smith¹, John B. Doe¹,²*
Affiliations:
¹ Laboratory of Regenerative Medicine, University of Science
² Department of Biomedical Engineering, Advanced Tech Institute
*Corresponding Author: [email protected]

1. Abstract

This protocol provides a detailed, reliable method for the isolation and expansion of human adipose-derived mesenchymal stem cells (AD-MSCs). The procedure involves the mechanical mincing and enzymatic digestion of lipoaspirate or adipose tissue samples, followed by filtration, centrifugation, and culture in a standard growth medium. This method consistently yields a high number of viable, multipotent MSCs that are positive for standard surface markers (CD73, CD90, CD105) and capable of trilineage differentiation. The standardized nature of this protocol makes it a valuable tool for applications in regenerative medicine, tissue engineering, and in vitro disease modeling, enhancing reproducibility across different laboratory settings.

2. Introduction

Mesenchymal stem cells (MSCs) are multipotent stromal cells with the capacity to differentiate into osteoblasts, adipocytes, and chondrocytes. While bone marrow was the traditional source, adipose tissue has emerged as an alternative, abundant, and easily accessible source of MSCs. Existing protocols for AD-MSC isolation can vary significantly, leading to inconsistencies in cell yield, purity, and functionality.

The protocol described here offers a standardized and visually demonstrated method that addresses key variables such as digestion time and tissue processing. The primary advantages of this method are its high cell yield, clear definition of critical steps, and the production of a cell population with consistent marker expression and differentiation potential, as confirmed by our representative results.

3. Materials and Equipment

3.1. Reagents and Chemicals

• Lipoaspirate or subcutaneous adipose tissue (from consenting donor)

• Phosphate-Buffered Saline (PBS), sterile

• Collagenase Type I (or similar enzyme blend, e.g., Collagenase NB 6)

• Bovine Serum Albumin (BSA)

• Dulbecco's Modified Eagle Medium (DMEM) - Low Glucose

• Fetal Bovine Serum (FBS)

• Penicillin-Streptomycin (P/S)

• Trypan Blue solution (0.4%)

• Trypsin-EDTA (0.25%)

3.2. Solutions Preparation

• Digestion Solution: 1 mg/mL Collagenase Type I in PBS. Filter sterilize using a 0.22 µm filter. Pre-warm to 37°C before use.

• Complete Culture Medium: DMEM supplemented with 10% FBS and 1% P/S.

3.3. Equipment and Labware

• Biological Safety Cabinet (BSC)

• CO₂ Incubator (set to 37°C, 5% CO₂)

• Centrifuge

• Water Bath or shaking incubator (37°C)

• 100 µm and 40 µm cell strainers

• Sterile surgical tools (scalpel, scissors, forceps)

• T-75 or T-175 culture flasks

• Sterile centrifuge tubes (15 mL and 50 mL)

• Hemocytometer or automated cell counter

4. Step-by-Step Procedure

Note: Perform all steps under sterile conditions in a BSC unless otherwise specified.

1. Tissue Washing and Mincing:

   1. Transfer the adipose tissue (approximately 10-20 mL) to a sterile Petri dish.

   2. Wash the tissue 3-4 times with an equal volume of PBS to remove red blood cells and debris.

   3. Using sterile scissors and forceps, meticulously mince the tissue into a fine slurry with fragments of roughly 1-2 mm³.

   4. Transfer the minced tissue to a 50 mL centrifuge tube.

   • Critical Step: Thorough washing and mincing are crucial for efficient enzymatic digestion.

2. Enzymatic Digestion:

   1. Add 2-3 volumes of pre-warmed Digestion Solution to the minced tissue.

   2. Cap the tube tightly and incubate in a 37°C water bath or shaking incubator for 45-60 minutes. Agitate the tube vigorously every 15 minutes.

   3. The digestion is complete when the solution becomes cloudy and the tissue fragments are largely dissociated.

3. Filtration and Cell Harvesting:

   1. Neutralize the digestion reaction by adding an equal volume of Complete Culture Medium containing FBS.

   2. Filter the cell suspension sequentially through a 100 µm cell strainer followed by a 40 µm cell strainer into a new 50 mL tube to remove undigested tissue and debris.

   3. Centrifuge the filtrate at 500 x g for 10 minutes at room temperature.

4. Cell Seeding and Culture:

   1. Carefully aspirate the supernatant, which contains adipocytes and the digestion solution.

   2. Resuspend the cell pellet (the Stromal Vascular Fraction or SVF) in 10 mL of Complete Culture Medium.

   3. Perform a cell count using a hemocytometer and Trypan Blue exclusion to assess viability and concentration.

   4. Seed the cells at a density of 5,000 - 10,000 cells/cm² in a T-75 culture flask containing 10-15 mL of pre-warmed Complete Culture Medium.

   5. Place the flask in a 37°C, 5% CO₂ incubator.

5. Medium Changes and Subculturing:

   1. After 48 hours, carefully remove the medium (non-adherent cells) and add fresh, pre-warmed Complete Culture Medium.

   2. Thereafter, replace the medium every 2-3 days.

   3. When cultures reach 70-80% confluence (typically after 5-7 days), passage the cells using 0.25% Trypsin-EDTA.

5. Representative Results

When performed successfully, this protocol should yield the following results:

• Cell Yield and Viability: Initial isolation typically yields 5 x 10⁵ to 1 x 10⁶ viable nucleated cells per gram of adipose tissue, with viability >95% as determined by Trypan Blue exclusion (See Video, Part 3).

• Cell Morphology: After 24-48 hours, adherent cells with a spindle-shaped, fibroblast-like morphology should be visible. These cells will proliferate and form colonies (Figure 1A).

• Surface Marker Expression: Flow cytometry analysis of passage 3 cells should show high expression (>95%) of typical MSC markers (CD73, CD90, CD105) and low expression (<2%) of hematopoietic markers (CD34, CD45, HLA-DR) (Figure 1B).

• Trilineage Differentiation: Under specific differentiation conditions, the cells should successfully differentiate into osteocytes (confirmed by Alizarin Red S staining), adipocytes (confirmed by Oil Red O staining), and chondrocytes (confirmed by Alcian Blue staining) (Figure 1C).

Figure 1: Representative Results of AD-MSC Isolation and Characterization.
(A) Phase-contrast image of adherent, spindle-shaped AD-MSCs at passage 2 (Scale bar: 100 µm).
(B) Flow cytometry histogram showing positive expression of MSC markers (CD73, CD90, CD105) and negative expression for hematopoietic markers (CD34, CD45).
(C) Trilineage differentiation potential: Osteogenesis (Alizarin Red S), Adipogenesis (Oil Red O), and Chondrogenesis (Alcian Blue).

6. Discussion

This protocol provides a robust and standardized method for isolating and culturing AD-MSCs. The key factors for success are the quality of the starting tissue, precise control of the digestion time to avoid over- or under-digestion, and the maintenance of strict sterile techniques throughout the process.

Potential modifications include adjusting the collagenase concentration or digestion time for different tissue sources or using different serum-free media formulations. The primary limitation is the potential for donor-to-donor variability in cell yield and growth kinetics.

This method is applicable for generating AD-MSCs for a wide range of downstream applications, including but not limited to, in vitro differentiation studies, 3D bioprinting, co-culture systems, and pre-clinical regenerative therapy research.

7. References

1. Bourin, P., et al. (2013). Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT). Cytotherapy, 15(6), 641-648.

2. Zuk, P. A., et al. (2001). Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Engineering, 7(2), 211-228.

Acknowledgments: We thank the members of the Laboratory of Regenerative Medicine for their technical support. This work was supported by [Funding Agency, Grant Number].
Video Correlation: The procedures and results described in this manuscript are visually demonstrated in the accompanying video.
Conflict of Interest: The authors declare no conflict of interest.