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.

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 the 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.

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.

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

In recent years, MSCs have been introduced as respectable candidates for regenerative medicine due to their pro-angiogenic, anti-apoptotic, and immunomodulatory attributes. A variety of human tissues can be used as a source of mesenchymal stem/stromal cells (MSCs), ranging from bone marrow (BM) to umbilical cord (UC). These cells are typically multipotent and can differentiate into a variety of cell types. MSCs have been studied extensively for potential applications in cardiomyopathy, neurodegenerative disorders, spinal cord injuries (SCI), kidney injuries, liver injuries, lung injuries, and even cancer. According to current research, MSC-derived extracellular vesicles (EVs) contribute to MSC-exerted therapeutic benefits.

As defined by the International Society for Extracellular Vesicles (ISEV), EVs are lipid bilayer particles secreted by cells that do not replicate. EVs can be categorised into three subclasses based on size and biogenesis procedures: surrounding exosomes (50-150 nm), microvesicles (MVs) (100-1000 nm), and apoptotic bodies (ApoBDs) (500-5000 nm). In order for exosomes to be produced, multiple steps must occur; endosomes must be created from the plasma membrane, intraluminal vesicles must be formed within multivesicular bodies by inward budding, the MVB must merge with the plasma membrane, and finally the internal vesicles must be released.

By transmitting their molecules, such as proteins, messenger RNA (mRNA), and microRNAs (miRNAs), MSC exosomes stimulate phenotypic changes and subsequently modify regenerative programs of target organs. A number of mechanisms are involved in phenotypic alterations, including prevention of apoptosis, cell proliferation, immunomodulatory reactions, attenuation of oxidative stress, and improving oxygen supply to recipient cells. By supporting mitochondrial transfer, MSC-exosomes can suppress inflammatory cytokine production and induce phenotype 2 alveolar macrophages (M2), leading to acute lung injury (ALI) rescue. It has been demonstrated that the transmission of miRNAs from MSC-exosomes to recipient cells is responsible for the restoration of damaged kidneys, hearts, livers, and brains

Various cells continuously form and secrete exosomes, including lymphocytes, platelets, mast cells, intestinal epithelium, dendritic cells, neoplastic cell lines, microglia, neurons, and MSCs. Studies have shown that exosomes play an important role in cell-to-cell communication as well as several physiological and pathological processes. Despite their inherent biological activities, exosomes have recently been introduced as encouraging drug carriers because of their small size, high biocompatibility, and ability to hold different therapeutic ingredients, including proteins, nucleic acids, and small molecules. There have been reports showing the usefulness of MSCs-exosomes for treating a variety of ailments, such as lung, kidney, liver, neurodegenerative, cardiac, and musculoskeletal diseases, as well as skin wounds in vivo.

As well as their remarkable therapeutic effects, MSC-EVs derived from diverse sources also possess a variety of physiological functions that may affect their therapeutic application. In a wide range of human disorders, MSC-exosomes are considered an effective alternative to whole-cell therapy because of their low immunogenicity and improved safety profile. Although MSC-exosome applications still face various challenges, their benefits and capabilities are attracting increasing interest.

To learn more about exosomes and their clinical applications or if you’re interested in boosting your clinical practice with exosomes, go to our website www.cellgenic.com

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

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

What are culture expanded Mesenchymal Stem cells?

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

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

Mesenchymal stem cells are expanded in a culture media, on a large scale in order to obtain the required quantity of cells needed for cellular therapy.

Culture expanded MSCs: How does it work?

Expanding Mesenchymal stem cells in a media involves step by step process of isolation and expansion.

Mesenchymal Stem Cells Isolation

Mesenchymal stem cells 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.

Mesenchymal stem cells are expanded in culture to increase their yield and amplify their desired functions and potency.

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

· Acquire fresh tissue extracts in strictly aseptic conditions, to maintain purity.

· To remove any cell clusters, you have to filter the cell suspension with a 70-mm filter mesh

· Use a centrifuge to roll the cells for about 5 minutes at 500g

·  Suspend the cells again the cells to measure the cell viability and yield using Trypan blue exclusion

· Use in T75 culture dishes to culture the cells in 10 mL of complete MSC medium at a density of 25 × 10⁶ cells/mL. You can then go on to Incubate the plates at 37 °C with 5% CO₂ in a humidified chamber without any interruption.

· When it’s past 3 h, remove the non-adherent cells that accumulate on the surface of the dish by changing the medium and replacing it with 10 mL fresh complete medium.

·  After an additional 8 h of culture, add 10ml fresh complete medium as a replacement for the existing medium. You’ll have to repeat this step every 8 h for up to 72 h of initial culture.

· Cells can be frozen in MSC growth media plus 10% DMSO (D2650) at a density of 2X10⁶ cells/vial.

Expansion of Mesenchymal Stem Cells in a culture media

Culture expanded mesenchymal cells undergo various stages from the preparation of the culture plate, thawing of Mesenchymal stem cells, and the actual expansion of Mesenchymal stem cells.

The reason behind the cultural expansion of Mesenchymal stem cells is to get them to differentiate into other cell types such as osteoblast, adipocyte, and mesenchymal stromal cells.

In preparation, to expand MSCs in a culture media, you need a 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.

The next step involves the thawing of the Mesenchymal stem cells, and here are a few steps for you to follow:

After the recommended culture medium and coated culture ware is ready and on standby, remove the vial of Mesenchymal Stem Cells from liquid nitrogen and incubate in a 37C water bath and pay close attention to it, until all the cells are completely thawed. The extent of completely thawed frozen cells and how fast, are what determines the cell viability.

Once the cells have thawed completely, take steps to avoid contamination by disinfecting the walls with 70% ethanol, before you proceed to the next step.

Place the cells in a hood, and carefully transfer the cells to a sterile tube with a pipette (1 or 2ml pipette), Do this in such a way to prevent bubbles.

Then, add drops of Mesenchymal Stem cell expansion medium that have been pre-warmed to 37C to the tube containing the Mesenchymal stem cells.

Be careful to take your time when adding the medium to avoid osmotic shock which could lead to decreased viability.

Proceed to mix the suspension slowly by pipetting up and down two times while avoiding any bubbles.

Place the tube in a centrifuge and centrifuge the tube at 300 x g for 2-3 minutes to roll the cells, and you should not vortex the cells.

After this, then decant as much of the supernatant as possible. These steps are necessary to remove residual cryopreservative (DMSO).

Suspend the cells in a total volume of 10 mL of Mesenchymal Stem Cell Expansion Medium again or any alternative of choice, pre-warmed to 37 °C, containing freshly added 8 ng/mL FGF-2 (F0291).

The next step involves placing the cell suspension onto a 10-cm tissue culture plate or a T75 tissue culture flask.

Maintain the cells in a humidified incubator at 37 °C  with 5% CO₂.

The next day, exchange the medium with fresh Mesenchymal Stem Cell 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.

Isolate the cells when they are approximately 80% confluent, using Trypsin-EDTA and passaged further or frozen for later use.

Expansion of Mesenchymal Stem Cells

Once the cells are actively proliferating and have reached a confluence of approximately 80% (before 100%), you should subculture the cells.

Then remove the medium from the 10-cm tissue culture plate containing the confluent layer of human mesenchymal stem cells, carefully and apply 3-5 mL of Trypsin-EDTA Solution, before proceeding to incubate in a 37 °C incubator for 3-5 minutes.

Crosscheck the culture to see if all the cells are completely detached. Then, add 5 mL Mesenchymal Stem Cell Expansion Medium to the plate.

Swirl the plate mildly to mix the cell suspension. Transfer the separated/isolated cells to a 15 mL conical tube.

Centrifuge the tube at 300 x g for 3-5 minutes to pellet the cells.

Throw the supernatant away and apply 2 mL Mesenchymal Stem Cell Expansion Medium (pre-warmed to 37 °C) containing 8 ng/mL FGF-2 to the conical tube and completely suspend the cells again. Remember not to vortex the cells.

Then, use a hemocytometer to count the number of cells.

Plate the cells at a density of 5,000-6,000 cells per cm² into the appropriate flasks, plates, or wells in a Mesenchymal Stem Cell Expansion Medium containing 8 ng/mL FGF-2.

Cells can be frozen in MSC growth media plus 10% DMSO (D2650) at a density of 2X10⁶ cells/vial.

Functions of Culture Expanded MSCs

Mesenchymal stem cells are required to be expanded in order for them to be used clinically for therapeutic purposes.

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

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

Mesenchymal stem cells 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.