Cell therapy: What techniques are used?

Several laboratories in the health network participate in the preparation of grafts (cells transplanted during the transplant). The structure of activities may vary from one establishment to another.

Some laboratories perform all the necessary tests and manipulations while elsewhere, several laboratories are called upon to obtain the various necessary expertise.

In most cases, this involves characterizing and storing the product pending the time of its administration to the patient. The preparation of grafts is divided into three main stages, namely the identification of blood stem cells, the characterization of the graft and the storage. Certain specific manipulations are also sometimes required.

Identification of blood stem cells

All the cells of the body have on their surface structures associated with their own function and which, taken together, define a particular type of cell. This is also the case with blood stem cells. One of these structures, the CD34 antigen, is found on stem cells and is used to determine their number. Demonstrating the presence of the antigen on a cell is made possible by a molecule complementary to the antigen, which is called an antibody. By attaching a fluorescent substance to the antibody, it becomes a tool.

The binding of the fluorescent antibody to its specific antigen makes the cell visible when the latter is analyzed by flow cytometry . We thus manage to enumerate the number of cells targeted by the antibody contained in the sample. In the case of stem cell removal by apheresis, this test is used to determine whether or not the removal should take place.

Indeed, as the response of different patients to the drug mobilizing blood stem cells outside the bone marrow to the blood can vary, it is evaluated on a blood sample if the collection threshold is reached. Below this threshold, experience has shown us that stem cell harvesting is ineffective. This test is also used on all grafts and it forms the basis of their characterization.

Characterization of the graft

In addition to determining the number of stem and progenitor cells present, the tests carried out on the graft samples make it possible to ensure the quality of the product by establishing the total number and the viability of the cells, to confirm the absence of contaminants, to verify the concordance of the blood group with that entered in the file and to draw a portrait of the other cell populations present.

In some cases, a functional test is also performed to confirm the potential progenitor of stem cells, that is, to assess the extent to which cells will be able to reform a complete blood system.

Storage

In some cases, the graft may be infused to the patient immediately after characterization or within 24 to 72 hours of collection. In other circumstances, it may take up to a few weeks before the patient receives it. This is particularly the case with autografts, where after the stem cells are taken, the patient undergoes chemotherapy and radiotherapy to completely eliminate their cancer. In such a situation, to preserve the cells in good condition outside the body, they must be cryopreserved.

Cryopreservation consists of freezing cells under conditions where their function and viability are preserved. This technique is very delicate. The cells are placed in an environment as close as possible to their physiological environment. A cryoprotective agent is added to protect cells from the formation of ice crystals which will damage and even kill the cells during freezing.

The temperature is then lowered slowly and in a controlled manner until it reaches -90o C, then the grafts are transferred to freezers cooled with liquid nitrogen and kept at a temperature below -150o C until their use. This way of freezing and storing cellular products makes it possible to keep them in good condition for several years, even more than 10 years.

You will have understood that the graft is the equivalent of a “cellular medicine”. Due to its nature, it cannot undergo sterilization at the end of the preparation. All graft handling is therefore done in compliance with aseptic techniques: wearing protective clothing, working in a biological hood and in a closed environment are the basis of all treatments.

Specific manipulations

In addition to evaluation and cryopreservation techniques, cellular products are subject to several specific and complex manipulations. For example, in cases of transplantation of cells taken from the bone marrow where the patient’s blood group and that of his donor are incompatible, it is necessary to remove the red blood cells which are found in large quantities in the graft. We therefore proceed to an erythodepletion using a Cobe 2991.

This device makes it possible, in successive stages of centrifugation and washing, to remove the red blood cells and to concentrate the white blood cells. Some protocols require that only the stem cells be infused to the patient. In these situations, other cells in the blood system need to be removed. This is done by a technique called immunomagnetic selection. At the end of the procedure, we get more than 95% of stem cells in the cell product, whereas initially, stem cells make up about 1% of all graft cells.

For some transplants, the graft is umbilical cord blood. Umbilical cord blood is processed and stored in external cell banks. The frozen product is received and thawed in the laboratory before transporting it to the patient’s bedside for administration. Most of the time, thawing is followed by a washing step aimed at removing the freezing medium. In certain circumstances, where the patient has been transplanted, but the disease resurfaces (recurrence), it is possible to infuse not stem cells, but rather immune system cells that fight cancer.

It is necessary to adjust the number of cells to obtain a desired beneficial effect, namely the elimination of cancer cells without causing a deleterious side effect. In fact, these cells can also, under certain circumstances, destroy normal cells of the skin, liver, digestive system, etc.

The development of new treatments

The majority of patients are treated with standard treatments, but there are still some cases where these treatments are inadequate or insufficient. The patients then find themselves without any solution. Fortunately, some laboratories are working on the development of new treatments. This is the case of the cell therapy laboratory at Maisonneuve-Rosemont Hospital, directed by Dr. Denis Claude Roy.

Dr. Roy’s team has worked on a technology to eliminate certain cell populations in a transplant. Various applications of this technique are being studied, including the elimination of cancer cells or even donor cells causing rejection after a transplant. In this context, this technology makes it possible to transplant cells from a donor who is only partially compatible with a patient. This strategy makes it possible to find donors for patients who, in the current treatment setting, had none.

The principle behind this concept is to remove cells from the transplant that would destroy the patient’s cells, while retaining the cells that destroy cancer cells and fight infection. Phase I / II clinical studies have been completed and an international study is currently underway. Another therapeutic approach is also being developed. The latter consists in increasing the number (expansion) of stem cells available when they are in too small quantity and thus allowing patients who cannot be transplanted or at risk of complications due to lack of stem cells to be treated.

The development of this approach is in the enhancement stage. This consists, after having done the small-scale studies, of adapting the intervention with a view to its use in a clinical context. A clinical study is expected to take place over the next year. More broadly, the clinical use of blood stem cells is not limited to the treatment of cancers and other hematological disorders. For several years, blood stem cells have been used to “repair” or regenerate sick hearts. Following an infarction, part of the heart no longer contracts, the heart cells are destroyed.

From a small sample of bone marrow, the stem cells are enriched by immunomagnetic selection technique and are then infused into the patient by intracoronary injection or directly into the heart where they will help repair damaged parts. Cell action can either be direct by replacing dead cells, or indirect by forming new blood vessels.

The Maisonneuve-Rosemont Hospital is currently participating in two clinical protocols where the cells manipulated in the laboratory are then transferred to CHUM teams to be administered to patients. Before distributing the cellular products for their infusion to patients, the files are reviewed and we make sure that the product meets the quality criteria allowing its release.

The product must contain the number of cells corresponding to the medical prescription and be free of contaminants. The cells must also be viable and demonstrate their efficiency in producing blood cells. Cell application-specific tests can also supplement the information needed.

Cell therapy: The laboratories of tomorrow

The work relating to cell therapy indeed places certain requirements for laboratories. On the one hand, it requires facilities where environmental conditions are strictly controlled. On the other hand, the application of a rigorous quality program. In Montreal, the Maisonneuve-Rosemont Hospital laboratory has developed a quality program to monitor all aspects of the work carried out in the laboratory. This program is based on the requirements of regulatory and accreditation bodies. The highlights of this program include the use of internal quality controls and participation in quality external programs comparing the results obtained with those of reference laboratories and other centers for a given test. The program also provides for the validation of all equipment and all procedures, the verification or audit of all processes as well as the review of quality indicators and incidents or accidents by a committee responsible for quality. Training activities (one year of work is necessary before mastering the procedures) and continuous staff training are also included. In Quebec, the Maisonneuve-Rosemont Hospital laboratory is also registered with Health Canada and the FDA for the type of manipulations it performs. He must also meet the accreditation Canada requirements for biomedical laboratory work and is accredited by the Foundation for the Accrediation of Cell Therapy (FACT). This organization aims to ensure that cellular products are prepared according to the most demanding quality criteria and are therefore safe for patients. Beyond standards and procedures, the laboratory team must be multidisciplinary. At MaisonneuveRosemont Hospital, it is made up of:

  1. several trained and experienced medical technologists; knowledge of hematology and blood banking is an asset. T
  2. he work requires understanding of protocols and processes, judgment, great adaptability and flexibility;
  3. a technical coordinator who organizes daily work and actively participates in the development and implementation of clinical study protocols;
  4. a specialist in clinical activities who is responsible for maintaining the quality system;
  5. a scientific and administrative coordinator who is responsible for all activities, the implementation of the quality program and the management of the laboratory;
  6. a medical director who instills a vision and provides scientific and medical support to the activities.

The entire “quality” component is closely linked to the hematopoietic cell transplant program and is therefore overseen by the quality framework advisor. The activities carried out in the cell therapy laboratory as well as the regulatory structure surrounding these activities have repercussions within the medical biology program. In fact, according to Hélène Desjardins, clinical and administrative head of the medical biology program at HMR, “the link between research and current clinical applications requires that clinical laboratories respond effectively to changes in practice. The daily integration of the concept of quality improvement requires the addition of resources with more scientific expertise than in routine laboratories. In terms of management, this represents a great challenge, because from a diagnostic laboratory, we are becoming a therapeutic laboratory where the preparation of living drugs requires technical, scientific skills and great flexibility. ” Adding different job titles to a team also requires reorganizing tasks. The choice and training of personnel working in an environment governed by so many regulations also becomes crucial, especially since cell therapy is not part of the basic training of medical technologists.

From the point of view of the clinical and administrative head, resources must be drawn from related sectors. Fortunately, she says, “such a change creates a quality dynamic and these new ways of doing things have a positive impact on all the other departments of medical biology”.