Optimize Microfluidics-Based Single Cell Isolation Workflow PDF
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Uploaded by FondRhenium
Steve Wiltgen, PhD
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Summary
This document, a presentation on microfluidics-based single-cell isolation, describes a workflow optimized for high efficiency, viability, and assurance of monoclonality in cell line development. The document highlights techniques such as limiting dilution and flow cytometry, explaining their respective advantages and disadvantages in achieving high-quality monoclonal antibody production. It also outlines an efficient procedure using a single-cell printer and its advantages compared to traditional methods.
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# A Microfluidics-Based Single Cell Isolation Workflow Optimized for Efficiency, Viability, and Assurance of Monoclonality ## Cell Line Development Workflow **Overall Goal:** Find the rare cell line that produces a large quantity of a high-quality product for a long period of time. | Workflow Step...
# A Microfluidics-Based Single Cell Isolation Workflow Optimized for Efficiency, Viability, and Assurance of Monoclonality ## Cell Line Development Workflow **Overall Goal:** Find the rare cell line that produces a large quantity of a high-quality product for a long period of time. | Workflow Step | Description | # of Clones | | ------------------------------- | ------------------------------------------------------------------------------------------------------------------------------- | ---------- | | Vector Construction & Transfection | Introduce DNA that encodes a monoclonal antibody into a cell line (typically CHO). | 1,000,000 | | Single Cell Isolation | Isolate a single CHO cell so that the product is monoclonal. Required by FDA because it impacts product quality. | 10,000 | | Monoclonality Verification | Provide evidence of monoclonality to the FDA, which usually includes statistical analysis and images. | 100 | | Select/Screen for Production | Find the CHO cells that are producing large quantities of the monoclonal antibody. | 100 | | Screen for Quality | Find the CHO cells that are producing high-quality monoclonal antibodies | 10 | | Clone Stability Testing | Find the CHO cells that can produce the monoclonal antibody for a long period of time. | 10 | ## Molecular Devices Product Portfolio for Cell Line Development **1,000's of Clones** - Limiting Dilution - Flow Cytometry - CloneSelect Imager - SpectraMax iD3 microplate reader _Well-established workflows for establishing clonal cell lines and assessing productivity._ **10,000's of Clones** - ClonePix 2 Colony Picker ## Principles of Single Cell Isolation ### Limiting Dilution (LD) - With LD, cells are diluted to a low concentration and are then deposited into a microplate. - Probability of clonality is governed by a Poisson Distribution. ### Flow Cytometry - With flow cytometry, cells are encapsulated by droplets in a process that is also governed by a Poisson Distribution. - Except that cell-containing droplets can be screened for single cells, thereby improving the likelihood of isolating a single cell per well. ## Single Cell Isolation Efficiency * **Limiting Dilution*:** Based on predicted values assuming 0.5 cells/well using LD. * **Flow Cytometry*:** Based on predicted values assuming 99% efficiency using flow. - Limiting Dilution: Low Efficiency (because it solely depends on Poisson distribution). - Flow Cytometry: High Efficiency (because of sorting prior to deposition). ## Probability of Monoclonality * **Limiting Dilution***:** Based on predicted values assuming 0.5 cells/well using LD. * **Flow Cytometry*:** Based on predicted values assuming 99% efficiency using flow. - Limiting Dilution: Depends on the accuracy of the cell counter and the accuracy of pipettes. - Flow Cytometry: Depends on gating parameters, the size of the nozzle, the frequency of droplet formation, the event rate and the heterogeneity of the cell population. ### Assurance of Clonality - Provided by capturing images of single cells. ## Single Cell Viability * **Limiting Dilution:** Manual or automated liquid handler. * **Flow Cytometry:** Flow sorting. - LD is a gentle method of handling cells. - Flow cytometry is stressful because of high pressures and shear stress. ## Typical Single Cell Cloning Efficiencies and Viabilities * **Limiting Dilution***:** Based on predicted values assuming 0.5 cells/well and 80% viability using LD. * **Flow Cytometry*:** Based on predicted values assuming 99% efficiency and 25% viability using flow. - Limiting Dilution: Low Efficiency (because it solely depends on Poisson distribution) and High viability (because of gentle cell handling). - Flow Cytometry: High Efficiency (because of screening prior to deposition) and low viability (because of the stressful environment required by flow). ## Low Efficiency of Clonal Outgrowth * **Limiting Dilution***:** Based on predicted values assuming 0.5 cells/well and 80% viability using LD. * **Flow Cytometry*:** Based on predicted values assuming 99% efficiency and 25% viability using flow. - In terms of clonal outgrowth, both techniques are fairly similar in their inefficiencies: - LD = 24% - Flow = 25% ## Molecular Devices Product Portfolio for Cell Line Development **1,000's of Clones** - CloneSelect™ Single-Cell Printer™ - CloneSelect™ Imager - SpectraMax® iD3 microplate reader _Well-established workflows for establishing clonal cell lines and assessing productivity._ **10,000's of Clones** - ClonePix™ 2 Colony Picker ## CloneSelect™ Single-Cell Printer™ - Microfluidics-based technology (similar to an inkjet printer). - Uses real-time image analysis to sort and deposits single cells into standard 96- or 384-well microplates with high efficiency and high viability. - Simultaneously provides image evidence of monoclonality. ## How CloneSelect SCP Works - Droplet generated by SCP printing cartridge. - Image of droplet captured. - Software automatically identifies the presence of cells. - If no cell or two cells are present, the droplet is siphoned away by vacuum. - If a single cell is present, the vacuum is turned off and the cell is deposited in the microplate. - Move to the next well and repeat. ## Single Cell Isolation * **Single-Cell Printer** - Just like LD and flow, the isolation of single cells using the SCP follows a Poisson distribution. - Just like flow, cell droplets are screened prior to deposition. - Unlike flow, which determines the presence of single cells indirectly, the SCP uses imaging to directly measure cell number. ## Single Cell Cloning Efficiency * **Limiting Dilution***:** Based on predicted values assuming 0.5 cells/well and 80% viability using LD. * **Flow Cytometry*:** Based on predicted values assuming 99% efficiency and 25% viability using flow. * **Single-Cell Printer***:** Assumes 85% efficiency - **SCP:** Higher efficiency (>80%) than LD because of image screening. ## Single Cell Viability - Disposable cartridge with gentle microfluidics technology keeps cells viable. - Simply load cells at a concentration between 0.5–1 x 106 cells/mL. - Holds up to 80 µL cell sample and is refillable. ## Single Cell Cloning Efficiency and Viability * **Limiting Dilution***:** Based on predicted values assuming 0.5 cells/well and 80% viability using LD. * **Flow Cytometry*:** Based on predicted values assuming 99% efficiency and 25% viability using flow. * **Single-Cell Printer***:** Assumes 85% efficiency and 75% viability - **SCP:** Higher efficiency (>80%) than LD, higher viability (~75%) than flow, and a higher overall clonal outgrowth (~2.5x greater) than LD (24%) or Flow(25%). - **SCP = 65%** ## SCP vs. LD Data on Efficiency - SCP Concentration = 1 x 105 cells/mL. - Average single cell deposit efficiency of 88% (n=10, 96-well plates). - LD = 0.5 cells/well. - Average single cell deposit efficiency of 32% (n=10, 96-well plates). _With LD, theoretical numbers don't match experimental numbers. This discrepancy reduces confidence in the probability of monoclonality._ ## Assurance of Monoclonality * **Limiting Dilution:** Proportion of wells with a single cell is low. _Without an image, the probability of monoclonality is low._ * **Limiting Dilution + Imager:** Proportion of wells with a single cell is low. _Same method of isolation, but now probability is high because clonality is validated by directly measuring with an image_. ## High Assurance of Monoclonality with the SCP - Visual validation of single cell deposit with multiple images per well. - 10x magnification for single cell resolution. - >99% of images in focus. ## High Probability of Clonality Validated with CloneSelect™ Imager (CSI) - Based on data presented earlier, P (clonality) on the SCP = ~93%. - But with high assurance, >99% correlation between images captured on the SCP and the CSI. - Combine imaging on the SCP with imaging on the CSI. - Two independent, direct measurements of clonality. - High probability and assurance of clonality. - Subsequently screen clones for viability and morphology. ## Confirming Monoclonality Takes Much Less Time - Screen for clonality earlier in the workflow (when 100's of clones still in pipeline) using the Single-Cell Printer. - Confirm clonality with the CloneSelect Imager later in the workflow (when 10's of clones still in pipeline). ## Reduce Your Workload While Also Reducing Costs and Waste **Time to Screen 1,000 Viable Single Cells** | Method | # of Plates | Sample/Instrument Prep Time | Time to Deposit Per Plate | Time to Image (2 Minutes Per Plate) | Total Time (Minutes) | |---|---|---|---|---|---| | LD | 67 | 30 Minutes | 1 Minute | 134 Minutes | 231 | | FACS | 42 | 2 Hours | 5 Minutes | 84 Minutes | 414 | | SCP | 18 | 15 Minutes | 7.5 Minutes | 36 Minutes | 186 | ## High Rate of Viability (Comparable to LD) - Average cell viability of 75% across many cell lines tested. - Can vary depending upon cell line, media, gating, etc. - Validated across several cell types (CHO, HEK, hybridoma, etc). ## SCP Can Recover Viable Clones From a Population With Low Viability - CHO-S loaded with 1 µM Calcein-AM. - LD (at 0.5 cells/well) or SCP used to deposit into 384-well plate. - CSI-FL (CSI with optional fluorescence) used to acquire images. - Software automatically locates cells and calculates the number per well. ## SCP Showed Higher Viability Post-Deposit Compared to Limiting Dilution - Clone Viability of CHO cells labeled with Calcein-AM. - LD: 59% - SCP: 77% - n=3, 384-well plates ## Improved Viability Over LD May Be Due to Screening Criteria - Screening for cell viability achieved by sorting cells based on size and roundness. - Data presented at CLD conference in Amsterdam, 2017 showed a similar result to our Calcein-AM experiment: - 91% viability with SCP vs. 68% for LD. ## Summary * **Limiting Dilution*:** Based on predicted values assuming 0.5 cells/well and 80% viability using LD. * **Single-Cell Printer*:** Assumes 85% efficiency and 75% viability. * **Flow Cytometry*:** Based on predicted values assuming 99% efficiency and 25% viability using flow. - High efficiency. High viability. High assurance of monoclonality. ## Molecular Devices Product Portfolio for Cell Line Development **1,000's of Clones** - CloneSelect™ Single-Cell Printer™ - CloneSelect™ Imager - SpectraMax® iD3 microplate reader _Well-established workflows for establishing clonal cell lines and assessing productivity._ **10,000's of Clones** - ClonePix™ 2 Colony Picker ## Molecular Devices Product Portfolio for Cell Line Development **1,000's of Clones** - CloneSelect™ Single-Cell Printer™ - CloneSelect™ Imager - SpectraMax® iD3 microplate reader - Octet HTX from ForteBio - Vi-Cell from Beckman Coulter _Well-established workflows for establishing clonal cell lines and assessing productivity._ **10,000's of Clones** - ClonePix™ 2 Colony Picker ## Molecular Devices Product Portfolio for Cell Line Development **1,000's of Clones** - CloneSelect™ Single-Cell Printer™ - CloneSelect™ Imager - SpectraMax® iD3 microplate reader - Octet HTX from ForteBio - Vi-Cell from Beckman Coulter _Well-established workflows for establishing clonal cell lines and assessing productivity._ **10,000's of Clones** - ClonePix™ 2 Colony Picker ## Using the SCP to Print Single Cells Onto Semi-Solid Media for Subsequent Screening on ClonePix - Day 0 images of single cells (CHOK1) printed in semi-solid media with 500 µm spacing. - Day 20 images of the same CHOK1 cells printed in semi-solid media.
