Nexcelom Bioscience
Adoptive Cell Transfer Therapy Cellometer Applications

Cellometer Use in Adoptive Cell Transfer Research

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Introduction

In recent years there has been a significant increase in new cancer immunotherapies. The Science journal called cancer immunotherapy, 2013 "Breakthrough of the Year" and Nature published an outlook article titled, "Calling Cell To Arms." These publications highlight the progression and growth of the cancer immunotherapy research field. Vaccine therapies, antibody therapies, immune checkpoint inhibitors, and adoptive cell transfer therapies are all strategies within the immunotherapy research field aimed at enhancing the immune system's ability to fight cancer.

Adoptive cell transfer (ACT) therapy focuses of using the patient's own cells for therapeutic treatment. Within ACT there are three major therapeutic approaches that use cells to directly target and stimulate the immune system: Tumor-infiltrating lymphocyte (TIL), T-cell receptor (TCR), and chimeric-antigen receptor (CAR) therapies. Although the approach to immune system stimulation is different from one therapy to the next, they all use and require primary, purified, and clinical samples. Whether plating cells for an ELISA or proliferation assays or working toward isolation of TCR from humanized mice, these samples are routinely processed and re-processed for downstream experiments. At each step, measurement of cell viability and concentration remains an important element in the immunotherapy therapy development process.

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Measuring Cell Concentration and Viability

Using Trypan Blue to Measure Cell Concentration and Viability

Simple, User-friendly Cellometer Procedure

Every Cellometer instrument is capable of performing the trypan blue exclusion method. After staining the cells with trypan blue, and loading the sample into the counting chamber slide, the Cellometer instrument automatically captures and analyzes multiple cell images. Within seconds, the result table displays the live, dead, and total cell concentrations, percent viability as well as the average cell size.

Using Cellometer Mini, Auto 1000, Auto T4, Auto 2000, K2 and Vision

Simple, User-friendly Procedure

1. Pipette 20 µl

2. Insert Slide

3. Select Assay & Click Count

4. Results in 30 seconds or Less!

Trypan Blue Protocol

  1. Dilute the stock (0.4 %) with PBS to 0.2 %.
  2. Filter the trypan blue with 0.2 micron filter
  3. Mix the cell suspension at 1:1 with 0.2 % trypan blue
  4. Load the counting chamber slide into the Cellometer and analyze

Using Acridine Orange/Propidium Iodide (AO/PI) to Measure Cell Concentration and Viability

Simple, User-friendly Cellometer Procedure

Dual fluorescence-based instruments (Vision, Auto2000, and K2) are capable of measuring the concentration and viability of nucleated cells in clinical samples without the need to perform red-blood cell lysis. Just 20 µl of sample is added to the Cellometer Counting Chamber. Imaging and analysis of the samples is completed in 30 seconds or less. Bright field and fluorescent cell images can be viewed to check cell morphology and verify cell counting. Total cell count, concentration, and mean diameter are automatically displayed for each counted sample.

Using Cellometer Vision, Auto 2000 and K2

AO/PI Protocol

  1. Obtain Nexcelom AO/PI solution: Cat # CS2-0106-5ML.
  2. Stain cell sample at 1:1 with AO/PI solution
  3. Load counting chamber slide and analyze
Pipette 20 µl of cell sample into a disposable counting slide

1. Pipette 20 µl of cell sample into a disposable counting slide.

Insert slide in to cell counter

2. Insert slide into the instrument

select AO/PI assay

3. Select assay from a drop down menu

cell count, concentration, and diameter

4. Click count, acquire image and view cell count, concentration, diameter

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Human and Animal Cells Counted by Cellometer that are used in Adoptive Cell Transfer Therapy

Human Melanoma Digest Sample Stained with AOPI

Human Melanoma Digest Sample Stained with AOPI

Human Melanoma Digest

First step in the isolation of tumor-infiltrating lymphocytes (TILs) is the excision and digestion of the tumor sample.

  • Use Cellometer to determine concentration and viability of TILs to plate for T-cell isolation post tumor digestion.
  • We recommend using AOPI to measure cell viability

Isolated T-cells Stained with AOPI

Isolated T-cells Stained with AOPI

Isolated T-cells

Secretion of IFNγ by TIL indicates tumor specific response by the isolated T-cells. An ELISA assay is performed to test for high levels of interferon-γ (IFNγ).

  • Use Cellometer to determine concentration and viability of T-cells needed for an ELISA assay.
  • We recommend using AOPI or TB to measure cell viability

Harvested Splenocytes Stained with AOPI

Harvested splenocytes stained with AOPI

Harvested Splenocytes

Immunize mice with tumor-specific antigen. Harvest mouse serum and splenocytes for B cell isolation

  • Use Cellometer to determine concentration and viability of harvested splenocytes
  • We recommend using AOPI to measure cell viability

Isolated B-cells Stained with AOPI

Isolated B-cells stained with AOPI

Isolated B-cells

Expand selected B-cell clone for hybridoma production.

  • Use Cellometer to determine concentration and viability of antibody producing hybridoma B-cells.
  • We recommend using AOPI or TB to measure cell viability

HEK 293 Cells Stained with Trypan Blue

HEK 293 cells stained with trypan blue

HEK 293 Cells

Using packaging cell line (HEK 293) for the production of the viral vector

  • Use the Cellometer to determine the concentration of cells needed to seed for viral vector production.
  • Use the Cellometer to determine the concentration of cells needed to seed for testing of the produced viral vector.
  • We recommend using AOPI or TB to measure cell viability

Human PBMCs Stained with AOPI

Human PBMCs stained with AOPI

Human PBMCs

Collect patient's peripheral blood lymphocytes for T-cell isolation.

  • Use the Cellometer to determine the viability and concentration of the collected PBMC sample.
  • Test sample concentration and viability post Leukapheresis + Ficoll gradient
  • We recommend using AOPI to measure cell viability

Isolated Human T-cells Stained with AOPI

Isolated Human T-cells stained with AOPI

Isolated Human T-cells Stained with Trypan Blue

Isolated Human T-cells Stained with Trypan Blue

Isolated Human T-cells

Patient's purified T-cells are often cryopreserved before they are transduced or transfected with a vector containing the cancer-specific t-cell receptor.

  • Use Cellometer to check the concentration and viability of cells before freeze-down
  • Use Cellometer to check the concentration and viability of samples after thawing
  • In preparation for transduction/transfection use the Cellometer to check the viability and determine optimal cell concentration for plating
  • Use the Cellometer to examine cell viability and concentration during cell expansion
  • We recommend using AOPI or TB to measure cell viability

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Table of Recommended Viability Detection Methods

The Cellometer line of instruments can quickly and accurately obtain the viability of the cell sample by trypan blue or fluorescent methods. Additionally, the system automatically reports an accurate cell count, concentration, and cell size in a single 20 µl assay. The fluorescent based Cellometer instruments (Auto 2000, Vision, and K2) also allow researcher to count the total number of nucleated cells in a fresh clinical sample without the need to lyse red blood cells. The table below outlines the recommended Cellometer instrument and assay use for each selected application.

Cell Type
Recommended Viability Detection Method
 
Trypan Blue
AOPI
Hybridoma cells
Yes
Yes
293T cells
Yes
Yes
T-cells for expansion
Yes
Yes
Tumor digest
Yes
Isolated B-cells
Yes
Yes
Splenocytes - Fresh
Yes
Splenocytes post RBC lysis
Yes
Yes
PBMC - isolated
Yes
Yes
PBMC - fresh
Yes
Feeder cells (K562 cells)
Yes
Yes

Cell concentration and viability measurements are performed in preparation for downstream assays such as ADCC, cytokine release, killing assays, ELISA.

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Assays Performed by Cellometer for Adoptive Cell Transfer Therapy

Proliferation Assay

Proliferation assay

Reprogramming CD19-Specific T Cells with IL-21 Signaling Can Improve Adoptive Immunotherapy of B-Lineage Malignancies. Harjeet Singh et al. Cancer Research; 71(10) May 15, 2011.


"The rate of T-cell growth cultured on γ-irradiated aAPC for 28 days in the presence of IL-2 (n=4) or IL-2 and IL-21 (n=7). The total number of CAR+ T-cells propagated over time."

"T cells were enumerated every 7 days and viable cells counted based on Trypan blue exclusion using Cellometer automated cell counter (Auto T4 Cell Counter, Nexcelom Bioscience). At the time of electroporation and during the course of coculture, programs "PBMC_human_frozen" and "activated T cell," respectively, were used for counting cells on the Cellometer."

Percent of GFP positive cells following transduction/transfection of HEK 293 cells

  • HEK 293 cells were cultured in flasks
  • Lentiviral vector containing an eGFP reporter was added to the cells at different concentrations: Control (no viral vector), Sample1, and Sample2
  • After an incubation period, the cells were collected and processed using a Cellometer Vision
  • Percent transduction = (Total GFP+ cells / Total bright-field cells) x 100

Sample 1

GFP positive cell sample 1

Sample 2

GFP positive cell sample 2

Control

GFP positive cell control
Lentivirus Transduction Total Cells GFP Positive % GFP Positive
Sample 1 1244 718 58.0%
Sample 2 1404 337 24.1%
Control 1188 0 0.00%

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Conclusions

The Cellometer instruments are capable of performing concentration, viability and cell size measurements of cultured and primary cell lines as well as perform more complicated cell based assays that are routinely used during the development of cancer immunotherapies. With bright-field and fluorescent platforms, these robust instruments are capable of performing consistent and accurate cell measurements for a large variety of samples. Dual fluorescence-based units (Auto 2000, K2, and Vision) are capable of measuring the concentration and viability of nucleated cells in clinical samples without the need to perform red-blood cell lysis. For isolated or purified cell samples the bright-field trypan blue Cellometers (Mini, Auto 1000, and T4) acquire multiple images, and automatically report the percent viability, cell concentration, and average mean diameter in under 30 seconds. Together these instruments cover every cell counting need that researchers may have in the immunotherapy field.

Give us a call Give us a call 978-327-5340. Experienced Nexcelom Applications Specialists are available 8:30am to 5:30pm EST to assist with selection of a Cellometer system.
Ask a Cellometer Specialist

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Publications Using Cellometer for Adoptive Cell Transfer Therapy Research

  1. Talebian L, Wu JY, Fischer DA, et al. (2012) Novel mobilization strategies to enhance autologous immune effector cells in multiple myeloma Front Biosci (Elite Ed) 3: 1500–1508
  2. Singh H, Figliola MJ, Dawson MJ, et al. (2011) Reprogramming CD19-specific T cells with IL-21 signaling can improve adoptive immunotherapy of B-lineage malignancies. Cancer Research 71(10): 3516-27
  3. Manuri PV, Wilson MH, Maiti SN, et al. (2010) piggyBac transposon/transposase system to generate CD19-specific T cells for the treatment of B-lineage malignancies. Human Gene Therapy 21(4):427-37.
Publications Citing Cellometer Instruments Used for Measuring Cell Viability with AOPI or TB »

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