Introduction to Atherosclerosis

Atherosclerosis is the most common form of arterial occlusive disease in adults. About 15 percent of adults over 55 years of age suffer from critical ischemia, the most severe form of this disease.

Due to the gradual aging of the population and the growing number of people in their third age group, a number of studies have been conducted in order to improve the prognosis of atherosclerosis obliterans and to find alternatives to the mutilation of the extremities. As a general rule, chronic ischemia of the lower limbs should be treated to alleviate symptoms, particularly pain, prevent disease progression, and reduce the rate of amputations. In most patients with critical ischemia, the main goal is to preserve the affected limb.

Atherosclerosis Obliterans Grade IV

The development of regenerative medicine is closely linked to the development of new knowledge about embryonic and adult stem cells, as well as the regenerative and therapeutic potential of stem cell therapy. The use of adult stem cells in the treatment of peripheral artery diseases has been demonstrated as a therapeutic agent for inducing angiogenesis. Recent preclinical studies, as well as pioneering clinical studies, indicate that bone marrow-derived mononuclear cells (MBMCs) can enhance tissue vascularization in ischemic limbs, with results similar to those obtained with peripheral blood stem cells supply.

Case Studies and Research

Cuba presented the first studies carried out in 2004 at the Institute of Hematology of the “Enrique Cabrera” hospital in Havana City, which achieved encouraging clinical results and had very few adverse effects in recent years.

A progressive rise in the accumulated experience with stem cells was also observed in Pinar del Rio in 2005, as the first 10 cases were carried out. The rising ease of obtaining this type of cell has made research and applications with these cells advance rapidly with great expectations in terms of clinical application.

Research Findings on Atherosclerosis Obliterans Grade IV

A study published by Dia-Diaz, et al. in the Journal of Medical Sciences of Pinar del Rio examined 296 patients with grade IV atherosclerosis obliterans between 2009 and 2019. During the study, autologous stem cells were injected intramuscularly from peripheral blood. Within four weeks, pain relief was observed, as well as an increase in the pain-free claudication distance. Angiography after treatment revealed collateral vessel formation. The limb was saved in 201 patients (68%), while 95 cases (32%) presented amputation criteria. Complications were not reported following the procedure.

The study demonstrated the effectiveness of the implantation of autologous stem cells obtained from peripheral blood, as well as the favorable evolution of patients, clinical improvement of rest pain, walking distance without claudication, and ankle-brachial pressure index.

We still need to explore a lot of ground in terms of these and other conditions. You can learn more about regenerative medicine and stem cells by enrolling in our international certification program at www.issca.us.

Mesenchymal Stem Cells (MSCs) are the most commonly used cells in stem cell therapy and regenerative medicine due to their high multi-potency. MSCs can be isolated from different tissues in the body.

In this article, you’ll learn about culture-expanded MSCs, how MSCs can be expanded, the potency of MSCs, and the types of cells they can differentiate into.

What Are Culture Expanded Mesenchymal Stem Cells?

Mesenchymal stem cells are highly potent cells used for cellular therapy and are isolated from different parts of the body. MSCs can be used to improve patient outcomes in diseases and conditions such as autoimmune diseases, degenerative diseases, nerve damage, diabetes mellitus, bone problems, etc.

For every patient, millions of MSCs are needed, and the exact amount varies according to disease, route of administration, administration frequency, weight, and age of the patient.

MSCs are expanded in a culture medium on a large scale to obtain the required quantity of cells needed for cellular therapy.

Culture Expanded MSCs: How Does It Work?

Expanding MSCs in a medium involves a step-by-step process of isolation and expansion.

Mesenchymal Stem Cells Isolation

MSCs can be isolated from different tissues in the human body such as adipose tissues, dental pulp, human bone marrow, umbilical cord tissue, umbilical cord blood, peripheral blood, and synovium.

MSCs are expanded in culture to increase their yield and amplify their desired functions and potency.

Although the population of MSCs obtained will vary from donor to donor, here are some steps to follow:

1. Acquire fresh tissue extracts in strictly aseptic conditions to maintain purity.
2. To remove any cell clusters, filter the cell suspension with a 70-mm filter mesh.
3. Use a centrifuge to roll the cells for about 5 minutes at 500g.
4. Suspend the cells again to measure the cell viability and yield using Trypan blue exclusion.
5. Use T75 culture dishes to culture the cells in 10 mL of complete MSC medium at a density of 25 × 10^6 cells/mL. Incubate the plates at 37°C with 5% CO2 in a humidified chamber without any interruption.
6. After 3 hours, remove the non-adherent cells by changing the medium and replacing it with 10 mL of fresh complete medium.
7. After an additional 8 hours of culture, add 10 mL of fresh complete medium as a replacement for the existing medium. Repeat this step every 8 hours for up to 72 hours of initial culture.
8. Cells can be frozen in MSC growth media plus 10% DMSO (D2650) at a density of 2X10^6 cells/vial.

Expansion of Mesenchymal Stem Cells in a Culture Medium

Culture-expanded MSCs undergo various stages from the preparation of the culture plate, thawing of MSCs, and the actual expansion of MSCs.

The reason behind the cultural expansion of MSCs is to get them to differentiate into other cell types such as osteoblasts, adipocytes, and mesenchymal stromal cells.

Preparation

To expand MSCs in a culture medium, you need culture ware. You can get one plastic or glassware plate and coat it with a sufficient amount of 0.1% gelatin. Don’t forget to aspirate the gelatin solution from the coated plate or flask before you use it.

Thawing of Mesenchymal Stem Cells

1. After the recommended culture medium and coated culture ware is ready and on standby, remove the vial of MSCs from liquid nitrogen and incubate in a 37°C water bath until all the cells are completely thawed. The extent of completely thawed frozen cells and how fast they thaw determines cell viability.
2. Once the cells have thawed completely, disinfect the walls with 70% ethanol before proceeding to the next step.
3. Place the cells in a hood, and carefully transfer the cells to a sterile tube with a pipette (1 or 2 mL pipette), doing this to prevent bubbles.
4. Add drops of MSC expansion medium that have been pre-warmed to 37°C to the tube containing the MSCs.
5. Mix the suspension slowly by pipetting up and down two times while avoiding any bubbles.
6. Place the tube in a centrifuge and centrifuge the tube at 300 x g for 2-3 minutes to roll the cells, avoiding vortexing the cells.
7. Decant as much of the supernatant as possible to remove residual cryopreservative (DMSO).
8. Suspend the cells in a total volume of 10 mL of MSC Expansion Medium again or any alternative of choice, pre-warmed to 37°C, containing freshly added 8 ng/mL FGF-2 (F0291).
9. Place the cell suspension onto a 10-cm tissue culture plate or a T75 tissue culture flask.
10. Maintain the cells in a humidified incubator at 37°C with 5% CO2.
11. The next day, exchange the medium with fresh MSC Expansion Medium (pre-warmed to 37°C) containing 8 ng/mL FGF-2. Replace with fresh medium containing FGF-2 every two to three days thereafter.
12. Isolate the cells when they are approximately 80% confluent, using Trypsin-EDTA, and passaged further or frozen for later use.

Functions of Culture Expanded MSCs

MSCs are required to be expanded in order for them to be used clinically for therapeutic purposes.

Culture-expanded MSCs can be induced to differentiate into adipocytes, osteocytes, hepatocytes, chondrocytes, tenocytes, and cardiomyocytes.

Due to their potential to differentiate into different kinds of cells in the body, MSCs can be used to manage liver problems, heart problems, joint and bone problems, etc.

MSCs are also used in tissue regeneration and modulation of the immune system. They possess anti-apoptotic, angiogenic, anti-fibrotic, and anti-oxidative properties.

However, the quantity of MSCs isolated from body tissues is not enough for clinical and therapeutic applications.

This is why MSCs are expanded in culture to increase their yield for desired therapeutic effect.