Background

As part of a project led by a leading pharmaceutical company developing a cell therapy product based on mesenchymal-derived neuro-like stem cells for the treatment of Alzheimer disease, the team that invented LyoLife™ was engaged to develop a freeze-dried product that did not contain DMSO showed >85% viability, and could be grown in culture.

Methodology

Full methodology is confidential and owned by the pharmaceutical company. For the freeze drying, we optimized the conditions of directional freezing and added the antioxidant epigallocatechin gallate (EGCG) to the freezing solution. After freeze-drying, we tested cell viability and growth rates.

Principal findings

Freeze-thawing results were excellent, having more than 90% viability and high growth rates. In addition, a device for freeze-drying larger volumes in cryo-bags was built (akaVTG). Large volumes of 50 ml after freeze-thawing showed the same viabilities and growth rates as small volumes when frozen without DMSO.

After freeze-drying, viability was approximately 70% and growth rates were slower (more time was required to reach 90% confluence). Samples were sent to the pharmaceutical company for evaluation and further experiments were performed within their labs to enable in-house functional evaluation.

Conclusions

Freeze-drying of human neuro-like stem cells derived from mesenchymal stem cells achieved 70% viability with demonstrated growth in culture, albeit at slower rates than freeze-thawing.

Source

Confidential research.

Supporting information

Figure 1. Post-thaw viabilities after freezing at different cooling rates using the VTG device compared to freezing with 10% DMSO using Mr. Frosty™ (freezing container filled with isopropyl alcohol at room temperature).

Figure 2. Cells viabilities (%) and remaining sample volumes (%) after different drying durations

Figure 3. Cell viability after drying for 6 hours

Cell concentration (∙106/ml) % Viability
Immediate rehydration 2.50 ± 0.10 46.40 ± 7.90
3 days at -80°C0 2.06 ± 0.05 47.00 ± 6.80
3 days at -20°C 2.10 ± 0.10 48.30 ± 6.20
3 days at -4°C 1.93 ± 0.05 47.40 ± 5.70

Table 1. Samples after 24 hours drying, followed by storage and rehydration with medium

Supporting information

Figure 1. Post-thaw viabilities after freezing at different cooling rates using the VTG device compared to freezing with 10% DMSO using Mr. Frosty™ (freezing container filled with isopropyl alcohol at room temperature).

Figure 2. Cells viabilities (%) and remaining sample volumes (%) after different drying durations

Figure 3. Cell viability after drying for 6 hours

Cell concentration (∙106/ml) % Viability
Immediate rehydration 2.50 ± 0.10 46.40 ± 7.90
3 days at -80°C0 2.06 ± 0.05 47.00 ± 6.80
3 days at -20°C 2.10 ± 0.10 48.30 ± 6.20
3 days at -4°C 1.93 ± 0.05 47.40 ± 5.70

Table 1. Samples after 24 hours drying, followed by storage and rehydration with medium

Supporting information

Figure 1. Post-thaw viabilities after freezing at different cooling rates using the VTG device compared to freezing with 10% DMSO using Mr. Frosty™ (freezing container filled with isopropyl alcohol at room temperature).

Figure 2. Cells viabilities (%) and remaining sample volumes (%) after different drying durations

Figure 3. Cell viability after drying for 6 hours

Cell concentration (∙106/ml) % Viability
Immediate rehydration 2.50 ± 0.10 46.40 ± 7.90
3 days at -80°C0 2.06 ± 0.05 47.00 ± 6.80
3 days at -20°C 2.10 ± 0.10 48.30 ± 6.20
3 days at -4°C 1.93 ± 0.05 47.40 ± 5.70

Table 1. Samples after 24 hours drying, followed by storage and rehydration with medium

Supporting information

Figure 1. Post-thaw viabilities after freezing at different cooling rates using the VTG device compared to freezing with 10% DMSO using Mr. Frosty™ (freezing container filled with isopropyl alcohol at room temperature).

Figure 2. Cells viabilities (%) and remaining sample volumes (%) after different drying durations

Figure 3. Cell viability after drying for 6 hours

Cell concentration (∙106/ml) % Viability
Immediate rehydration 2.50 ± 0.10 46.40 ± 7.90
3 days at -80°C0 2.06 ± 0.05 47.00 ± 6.80
3 days at -20°C 2.10 ± 0.10 48.30 ± 6.20
3 days at -4°C 1.93 ± 0.05 47.40 ± 5.70

Table 1. Samples after 24 hours drying, followed by storage and rehydration with medium

Supporting information

Figure 1. Post-thaw viabilities after freezing at different cooling rates using the VTG device compared to freezing with 10% DMSO using Mr. Frosty™ (freezing container filled with isopropyl alcohol at room temperature).

Figure 2. Cells viabilities (%) and remaining sample volumes (%) after different drying durations

Figure 3. Cell viability after drying for 6 hours

Cell concentration (∙106/ml) % Viability
Immediate rehydration 2.50 ± 0.10 46.40 ± 7.90
3 days at -80°C0 2.06 ± 0.05 47.00 ± 6.80
3 days at -20°C 2.10 ± 0.10 48.30 ± 6.20
3 days at -4°C 1.93 ± 0.05 47.40 ± 5.70

Table 1. Samples after 24 hours drying, followed by storage and rehydration with medium