The culture of primary cells is essential to many research and therapeutic applications. To access single cell suspensions for further research and cell culture, the first step is to start with a tissue sample. The tissue then needs to be processed to dissociate the cells so that they may be studied further. These primary cells are frequently used for biomedical research, cell biology studies, or for transplant and Cell Therapy applications. These individual cells can either be isolated into a primary cell culture or harvested together and replated for a secondary culture.
There are three primary methods for tissue dissociation and frequently the methods are combined. These include: enzymatic dissociation, chemical dissociation and mechanical dissociation. Enzymatic dissociation is the process of using enzymes to digest cut-up tissue pieces thereby releasing cells from tissue. Many different types of enzymes are used in this process and they can also be used in combination. Certain enzymes are more effective with certain tissue, so it is best to find the right enzyme or combination of enzymes for each specific tissue type.
Chemical dissociation takes advantage of the fact that cations participate in the maintenance of intracellular bonds and the intracellular matrix. By introducing EDTA or EGTA, which binds these cations, the intercellular bonds are disrupted.
Lastly, mechanical dissociation requires cutting, scraping or scratching the tissue into small pieces, then the minced up tissue is washed in medium in order to separate the cells from the tissue and sometimes gentle agitation is also used to help loosen the cells.
The selection of a method for dissociation is usually made based on the tissue type, tissue origin and what methods have been found to be most successful in the literature.
Tissue Dissociation Challenges
The goal of most tissue dissociation processes is to obtain the most viable cells possible and ensure that the method doesn’t have a negative impact on cell viability or function. Researchers must choose the right combination of dissociation methods in order to achieve success. Most rely on literature searches, but are often faced with conflicting advice. The reasons for the inconsistent data can often be attributed to varying protocols and inconsistent and/or impure enzymes. Inconsistencies can impact the ability for researchers to accurately interpret the data within their own experiments and can make replicating the experiment impossible for others. This variability in the isolation process can also negatively impact the use of these cells in transplant or Cell Therapy applications.
One way to address variability is to create a standardized protocol for the tissue dissociation. If you look at enzymatic dissociation as an example, there can be problems in determining the correct concentration of enzyme, the correct incubation time and the correct temperature; getting these factors wrong can cause cell damage or incomplete dissociation.
Furthermore, another important key to addressing variability is to look at the quality of the enzymes. Enzymes come in a wide range of quality including purity, grade (i.e. manufactured using good manufacturing practices GMP), enzymatic strength and source (i.e. animal-origin free). Variability among these products both across grades and within lots is a challenge for researchers. Variability frequently causes inconsistent or non-reproducible results.
The manufacturing guidelines for Cell Therapy products are still evolving but one current applicable reference is the USP 1046, which indicates the importance of qualifying raw materials used in manufacturing. So considering issues such as raw material origin, consistency, risk of contamination and whether the raw material was manufactured under GMP is important when planning a manufacturing process that will eventually be submitted to governmental agencies for approval. As a result there is a growing interest in Animal-origin free (AOF) and Good Manufacturing Practice (GMP) grade enzymes.
Need for Articlesized Tissue Dissociation Protocols for Clinical Applications
To tackle the problems of variability, many researchers are moving toward creating a standardized protocol using GMP and/or AOF raw materials for tissue dissociation in order to ensure consistent research results that are reproducible and to provide appropriate procedures for transplant and clinical Cell Therapy applications. AOF materials are preferred because they don’t carry the risk of possible contamination with adventitious agents such as animal based viruses or prions. For Cell Therapy applications, these protocols include a GMP process and GMP grade enzymes as cell therapies that are deemed “more than minimally manipulated,” will be regulated as a biological product by the FDA and thus will be required to be in compliance with GMP standards.
Two recent publications highlighted the efforts to create consistent protocols for transplant and Cell Therapy applications. I have provided a brief highlight of the papers here.
Islet Isolation from the Pancreas
The need for a standard GMP protocol was highlighted in a recent paper published in the journal Transplantation Direct titled, “The Choice of Enzyme for Human Pancreas Digestion is a Critical Factor for Increasing Success of Islet Isolation.” In the article the authors discuss their work in islet isolation and the importance of finding an effective and consistent method for isolating islets from the pancreas. To this end, they compared key islet parameters to assess overall islet quality post isolation comparing three enzymes, liberase HI (Roche Diagnostics), collagenase NB1 NP (SERVA Electrophoresis GmbH) and liberase Mammalian Tissue Free (MTF) C/T (Roche Diagnostics). For clarification, these enzymes are for further processing only and not for direct application in humans.
Islet Cells for Transplantation
Islet transplantation is another treatment option for Type 1 diabetes. In islet transplantation, islets are harvested from the pancreas of an organ donor and cells are then infused into the liver. According to the National Diabetes Information Clearinghouse, “Transplant patients typically receive two infusions with an average of 400,000 to 500,000 islets per infusion. Once implanted, the beta cells in these islets begin to make and release insulin.”
The study authors explained that initially liberase HI was used to isolate islets and its use led to an increase in the number of patients that could receive islet transplants. However its use was stopped in 2007 because of concerns of possible prion contamination from bovine sourced materials. Liberase HI was not highly purified and had serious issues with lot-to-lot variability, thus consistent islet isolation and long term storage were problems. Collegenase NB1 supplemented with neural protease (NP) has been used as an alternative to liberase HI and this allowed for transplanting to continue for clinical grade islet production.
Study authors used a perfusion apparatus and infused one of the three enzymes in the study, liberase HI, collagenase NB1 with NP or liberase MTF C/T. Then the pancreas was cut into pieces and put into a Ricordi’s digestion chamber. The digestion was deemed complete when 50% or more of the islets were free from the tissue.
What they discovered is that the digestion efficacy was significantly higher, the glucose stimulation index was higher and islets had the highest success rate of transplantation in diabetic mice when the liberase MTF C/T was used. In addition lot-to-lot variability was much less compared to the other two enzymes.
Isolation of Mesenchymal Stem Cells from Umbilical Cord Tissue
Another recent paper highlighted work toward development of a standardized GMP protocol for dissociation and banking of Mesenchymal stem cells from umbilical cord tissue. The paper, “A New Articlesized Clinical-grade Protocol for Banking Human Umbilical Cord Tissue Cells,” published in the journal Transfusion, describes how the authors created a reliable and efficient clinical-grade protocol for the isolation of mesenchymal stem cells from umbilical cord tissue utilizing a combination of enzymes and a semiautomatic dissociator.
Source of cells for Cell Therapy
Mesenchymal stem cells have been studied in clinical trials for over 10 years. In this time they have been tested for their therapeutic properties against many diseases and injuries. A handful of these treatments include, tissue injury, retinal disease, ALS (amyotrophic lateral sclerosis, MS (multiple sclerosis) and liver failure.
Mesenchymal stromal cells (MSCs) are desirable for Cell Therapy because they possess self-renewal capability and multilineage differentiation into mesoderm ectodermic and endodermic cells. While MSCs can be found in most tissue they were originally derived from bone marrow. Now they are being isolated from many types of tissues, including adipose, muscle, dental pulp, placental and umbilical cord. MSCs are also relatively easy to expand in culture. The paper highlights some positive attributes of MSCs isolated from UCT including that tissue is easily sourced and cells exhibit a faster proliferation rate. The paper also states, “UCT-MSCs present many advantages compared to MSCs isolated from other adult tissues, due to the noninvasive collection procedure, their higher proliferation ability, their low immnogenicity, the multilineage differentiation ability, and their immunomodulatory and trophic properties.”
Because of the attractive attributes of using MSCs in Cell Therapy, there is a need to isolate these cells in a way that is consistent with the requirements for therapeutic applications. The authors of the paper set out to create a clinical grade isolation protocol for isolating MSCs from UCT. The desire was to create this protocol using GMP material but also keeping in mind that the protocol still needed to maintain isolation and cryopreservation techniques that created the best opportunity for good yield and quality of the isolated cells.
In the study the authors tested two strategies. The first was manual dissociation and the second was semiautomatic dissociation using Miltenyi Bio’s GentleMACS dissociator. To begin all tissue was cut into small pieces and put into a solution containing PBS, GMP grade collagenase NB6, and hyaluronidase (a medical product). In the manual dissociation method the tissue and solution were incubated for 2.5 hours with no agitation. In the semiautomatic dissociation, the sample was first mechanically dissociated with GentleMACS then incubated for 1.5 hours in solution.
When comparing the two methods, authors found that by using a combination of semiautomatic dissociation with enzymes that the isolation took less than half the time of using manual dissociation alone, which is an important time and labor savings when banking large numbers of cells. The process also produced cells that had the same features as the cells isolated via manual dissociation.
These two papers demonstrate both the need for and the successful implementation of a standardized clinical grade protocol for isolating cells from tissue samples. Current Cell Therapy manufacturing guidances indicate that reduction of variability, the need for good manufacturing practices and the use of animal-free origin material with regard to tissue dissociation will continue to increase in importance as therapies move toward commercialization. As such, it is important to share these protocols through publication so that others may benefit from the experience and so that over time, protocols continue to become more standardized.