Precision in PBMC Isolation and Analysis: Driving Next-Gen Therapeutic Solutions

This blog post was written by Mediford Corporation, a CRO of PHC Group providing comprehensive services for drug development including advanced treatment options, covering both non-clinical and clinical phases. Their services are available on the Scientist.com marketplace.

In recent drug developments, immunological knowledge has been applied to cancer immunotherapy, autoimmune diseases, infectious diseases and more. In these fields, understanding changes in the immune system after drug administration is crucial. Therefore, Peripheral Blood Mononuclear Cells (PBMCs) are often used to examine immune cell responses or phenotypes, which is particularly important for these studies.

PBMCs are composed of various immune cells such as T-cells, B-cells, NK cells, monocytes and dendritic cells. PBMCs are useful for evaluation of rare cells, including antigen-specific T cells, as red blood cells and granulocytes, which account for most of the blood, are removed, and long-term storage is possible by freezing. Due to these characteristics, they have been utilized in a variety of applications such as immunology research and vaccine development.

PBMCs are generally isolated and collected from fresh blood using density gradient centrifugation. Since the layering of blood onto a density gradient media and the collection and washing of PBMCs are done manually, skilled operational techniques are required to obtain high-quality and homogeneous PBMCs. Furthermore, in order to obtain pure PBMCs that more accurately reflect the in vivo biology, an operational system is necessary to carry out the process from blood collection to PBMC isolation in as little time as possible.

Mediford Corporation has been providing PBMC isolation services in clinical trials and clinical studies for almost 20 years. As soon as blood samples collected at medical institutions are delivered to our GLP-compliant laboratories in Tokyo and other locations in Japan, our certified and skilled researchers perform PBMC isolation. In addition to standard isolation methods, other isolation methods using commercial products such as Vacutainer CPT (BD), Leucosep tube (Greiner) and SepMate (STEMCELL Technologies) are available as well. We also arrange global transportation of isolated PBMCs upon request.

PBMC isolation procedure using Ficoll density gradient centrifugation

From the isolated PBMCs, we conduct various analyses, such as immune cell responses and phenotyping, using a range of platforms, including Flow Cytometry, ELISpot assay and qPCR analysis at our laboratory. Below, we highlight some of the most important platforms among these:

Flow Cytometry

The immune cells that are measured by flow cytometry include the main T cells, such as helper T cells and cytotoxic T cells, B cells that produce antibodies, dendritic cells and macrophages that present antigens, other T cells such as regulatory T cells and Th17 cells, which are important in regulating the immune system, and killer cells such as NK cells, NKT cells, and γδT cells. Through the network created by these immune cells, tumor cells and virus-infected cells are recognized as foreign bodies and eliminated. Conversely, in cases of autoimmune diseases and allergies, an overactive immune system can be detrimental to the body. Therefore, in drug development it is important to utilize and control these immune system functions and to confirm the changes and effects of drugs on immune system functions.

Application to drug development: Flow cytometry is used in a wide range of applications in drug development, including confirming pharmacokinetics and pharmacodynamics, assessing the effects of administration and analyzing drug characteristics. For example, in the field of tumor immunology, immune cells such as CAR-T cells are themselves drugs and are used for the purposes of TK/PK (toxicokinetics/ pharmacokinetics). When developing a vaccine to prevent infectious diseases, it is necessary to confirm the induction of cellular immunity and, in the case of drugs that may act on the immune system, to confirm the effect on immune cell populations. For drugs whose mechanism of action involves the immune system, bioassays may be followed by flow cytometry.

High throughput vs. High parameter: These immune cells express a variety of cell surface and intracellular markers that characterize their cell population and state. To obtain a more detailed understanding of the immune system, it is necessary to measure as many markers as possible. On the other hand, when screening many samples or bioassays generated in clinical trials, high throughput may be required rather than the number of analytical parameters. Thus, a system that can meet the needs of both high throughput and high parameterization is required.

Conventional Flow Cytometry: To meet the wide range of flow cytometry needs required for pharmaceutical development, we have two types of flow cytometers. The first is FACSLyric (BD), a conventional flow cytometer that measures fluorescence and is capable of high-throughput measurement using a 96-well plate and detecting up to 12 colors. In addition to immunophenotyping using PBMCs or whole blood as materials, in recent years it has become possible to conduct testing to evaluate the effectiveness of vaccines by culturing PBMCs with antigens and evaluating cells that react specifically to the antigen using intracellular cytokine production as an indicator. In these conventional flow cytometry processes, compensation of the fluorescence spectral overlap is often a concern; however, our experienced staff can flexibly handle such challenges as they arise.

Mass Cytometry: The other is CyTOF XT (Standard BioTools), a mass cytometer that detects metal mass and can essentially measure up to about 50 parameters. Because there is no signal overlap, which is often a concern with conventional flow cytometers, it is possible to comprehensively analyze immune cells in PBMCs or whole blood in a single measurement (Figure 1). For comprehensive analysis of white blood cells, ready-made kits are available, and it is of course also possible to design a measurement panel with the desired combination of markers. As the number of parameters increases, the measurement results become more complex, taking more time to evaluate and potentially overlooking important results. In recent years, dimensionality reduction methods such as t-SNE and FlowSOM are often used to solve these issues, and we can also handle these analysis methods (Figure 2).

ELISpot Assay

Enzyme-Linked ImmunoSpot (ELISpot) assays are often used to assess immune cell responses, such as to confirm the efficacy of infectious disease prevention and antitumor vaccines, and the safety of gene therapy using viruses as vectors.

Mechanism of immune response: In the case of infectious disease prevention vaccines, the administered vaccine is taken up by antigen-presenting cells as an antigen, processed within the cells, and then broken down into peptide fragments that bind to MHC (major histocompatibility complex; also known as HLA (human leukocyte antigen)). After the MHC bound to the antigen peptide is transferred to the cell surface of the antigen-presenting cell, T cells having a T cell receptor that specifically recognizes this antigen peptide-MHC complex recognizes them and are activated. Activated T cells work to protect the body from viral attacks by producing cytokines such as IFN-γ, which have antiviral activity, proliferating and increasing in number to make it easier to eliminate virus-infected cells, and helping B cells produce antibodies. An important aspect of these steps is that there are numerous types of MHC, which differ from person to person, and the length and sequence of the peptides that bind to each MHC vary, resulting in different responses to viruses from person to person.

Assay procedure: The ELISpot assay is an assay method that utilizes the immune reaction described above. The method is outlined below (Figure 3).

1. PBMCs (containing antigen-presenting cells and immune cells such as T cells) are cultured for a certain period of time in the presence of antigen (which is often a peptide pool with overlapping amino acid sequences that make up the antigen). In this case, culture plates whose wells are pre-coated with antibodies to capture cytokines, are used.
2. When T cells that react specifically to an antigen are present in PBMC, they are activated via antigen-presenting cells and produce cytokines.
3. Cytokines released from the cells are captured by antibodies on the bottom of the well close to the cells. Undesired cytokines diffuse into the medium.
4. After washing off the cells, the cytokines trapped on the membrane are visualized by detection antibodies. This procedure allows antigen-specific cells to be spotted and detected by an ELISpot reader.

Fit-for-purpose solutions to immune response: In most cases, IFN-γ is detected as cytokine, but immune responses are divided into Th1 and Th2 depending on the type of cytokine. It is said that Th1 enhances cellular immunity, while Th2 enhances humoral immunity, and IFN-γ is classified as Th1. Therefore, in order to confirm the immune system reaction in more detail, it may be necessary to simultaneously measure cytokines classified as Th2, such as IL-4. Therefore, we have an ELISpot reader that can measure up to two types of cytokines simultaneously, and can meet such needs.

Although the effectiveness of vaccines can also be evaluated by measuring intracellular cytokines using flow cytometry as mentioned above, the ELISpot assay, which uses a 96-well plate and can obtain data in a short period of time, has the advantage of being high-throughput and is often used as a vaccine evaluation system. Each of these platforms has its own advantages and disadvantages, so they may be used differently depending on the research objectives, and in some cases both may be implemented.

The isolated PBMCs can be used not only in the measurement systems mentioned above, but also in bioassays, such as for measuring cytokines released into the culture supernatant in response to drug stimulation. In addition to singleplex measurements using conventional ELISA, it is also possible to perform multiplex measurements using electrochemiluminescence or the principle of flow cytometry using beads. Also, gene expression analysis of stimulated cells can be performed using microarrays, and the pharmacokinetic progression of CAR-T cells can be monitored by qPCR analysis.

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