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Stem cell expansion
Pluripotent stem cell expansion using either embryonic stem cells (ES) or induced pluripotent stem cells (iPSC) is a critical technique for producing cells used in disease modeling, drug discovery and therapeutic development. The expansion process involves the proliferation of ES/iPSCs to obtain sufficient quantities for research or translation to the clinic while maintaining the ES/iPSC’s pluripotency and differentiation capacity.
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Stem cell expansion

Pluripotent stem cell expansion using either embryonic stem cells (ES) or induced pluripotent stem cells (iPSC) is a critical technique for producing cells used in disease modeling, drug discovery and therapeutic development. The expansion process involves the proliferation of ES/iPSCs to obtain sufficient quantities for research or translation to the clinic while maintaining the ES/iPSC’s pluripotency and differentiation capacity.

ProblemSolutionProof

Problem

The culture of pluripotent stem cells, wether in an R&D tissue culture lab or a GMP cell manufacturing suite, is technically challenging. A highly trained scientist must exercise precise motor control when exchanging reagents and harvesting the cells, as well as make judgment decisions on when to initiate process steps (e.g. passaging). These challenges are compounded by stem cells themselves being in a dynamically unstable cell state, frequently suffering from loss of pluripotency, spontaneous differentiation, or cell death.


In plain English. You usually need someone with a PhD to move liquids between plastic containers for hours on end without making any mistakes.


This is undesirable due to:

  1. Inconsistency  - Human variability, from the angle at which a pipette gun is held to the velocity of ejection of liquid from the stripette to deciding “what confluence” looks like creates points for poor process repeatability and failure.
  2. ‍ Contamination - poor aseptic technique, or honest mistakes, can contaminate cultures setting you back weeks.‍
  3. The need for Sleep - human beings generally don’t perform at their best when feeding cells at 3am.‍
  4. Boredom - Smart human beings thrive on solving interesting problems. Not being a machine. We predict having your team perform repetitive cell culture workflows will have negative effects on staff retention.‍
  5. Costs - have you ever tried to grow and manage a cell culture team? Operational expenses, equipment, and salaries shoot up fast. Surprisingly fast.

Solution

Your technical team creates value when designing/analyzing experiments. Not when they are feeding cells on the weekends. Give your team their weekends back and elevate your science. Use Emmet.
‍

Emmet combines fluidics, mechanical actuation, in-line metabolic sensing and machine intelligence (not to jump on the AI bandwagon) to provide a fully automated solution for TC flask based ES/iPSC expansion: from seeding and media exchanges to passaging and harvesting.


Critically, Emmet delivers unparalleled precision and standardization with control over almost every process parameter. From dictating the tilt angle of cell culture vessels (within 0.1 degrees) to ensuring homogenous fluid distribution during seeding and media exchanges.

Proof

In a benchmarking study, Emmet expanded iPSCs as well as a trained stem cell scientist while reducing operator time by over 80% across the eleven-day hiPSC expansion workflow.

A single T25 on Emmet was seeded with 200,000 cells/cm2 in 5 ml mTeSRTM Plus (Stemcell Technologies). Media exchanges were performed daily. On day four, cells were passaged from one T25 flask into two T75 flasks (1:6 passage ratio). Daily media exchanges of 15 ml were performed. On day eight, cells were passaged from one T75 flasks into three T225 flasks (1:9 passage ratio). Daily media exchanges of 45 ml were performed. Cells were harvested from the three confluent T225 flasks on day eleven. All cell culture on Emmet were mirrored with a manual control.

Figure 1: hiPSC expansion in Emmet and manual control. A) Brightfield microscopy images of hiPSCs. B) Growth curves of hiPSCs grown on Emmet and in manual culture. C) Flow cytometry analysis of pluripotency markers (OCT4, SSEA4, SOX2, TRA-1-60).

hiPSCs expanded on Emmet retained normal colony morphology (figure 1A), and the number of hiPSCs produced by Emmet was consistent with the manual control throughout  (figure 1B). After harvesting from both Emmet and the manual control, hiPSCs were assessed for the expression of pluripotency markers using flow cytometry. hiPSCs expanded both in Emmet and in the manual control had similar expression levels of pluripotency markers OCT4, SOX2, SSEA4, and TRA-1-60 (figure 1C). This indicates that both expanded hiPSC populations retained their pluripotency.

Figure 2: Functional analysis of hiPSC differentiation capacity. A)Images of embryoid bodies formed from hiPSCs cultured manually and in Emmet. B) Gene expression analysis of the pluripotency (OCT4), mesoderm (T), ectoderm (NEUROD1, PAX6), and endoderm (FOXA2, SOX17) lineage markers.

In addition to flow, we performed functional analysis by putting hiPSCs harvested from Emmet and manual culture through an embryoid body (EB) formation protocol.

We were chuffed that hiPSCs cultured on Emmet and the manual control were both successfully differentiated into EBs (figure 2A). PCR with cDNA from both EBs was performed, indicating a reduced level of pluripotency markers (OCT4) compared to pluripotent hiPSCs. Both EB’s were positive for markers from all three germ layers: mesoderm (T), ectoderm (NEUROD1, PAX6), and endoderm (FOXA2, SOX17) (figure 2B).

These results indicate that both hiPSCs expanded on Emmet and the manual control maintained tri-lineage differentiation capacity.

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Figure 3: Operator time to complete hiPSC expansion.
A) total time to complete hiPSC expansion workflow on Emmet and the manual control.

Finally, logs were kept of operator time spent on manual and Emmet hiPSC expansion protocols, which were both performed by the same operator. The manual hiPSC expansion required 17.25 hours of time, compared to 3 hours of time for the Emmet hiPSC expansion, a reduction in total time needed of 82.6% (figure 3A).

Whether your protocol calls for twice weekly or twice hourly media exchanges, Emmet can standardize and automate the expansion of any hiPSC line and, more broadly, any cell culture.

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All experiments performed by Unicorn Biotechnologies scientists at Unicorn's Sheffield, UK R&D lab on an Emmet: Generation 1 instrument.

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Emmet is an intelligent machine that automates your TC flask cell culture workflows. With a unified hardware and software stack in a single bench-top instrument, Emmet brings physical AI to the tissue culture lab.
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Protocol development

Our team will onboard your cells, reagents and protocol to a Unicorn system and develop an optimized protocol or bioprocess for you at our R&D center.
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