**3. Conclusions**

• day 6–20: put the cells daily or every second day to 0.5–0.7 × 10<sup>6</sup>

.

• Re-seed cells when concentration exceeds 2.5 × 10<sup>6</sup>

with: 50 ng/ml TPO and 10 ng/ml IL-1β at 0.5 × 10<sup>6</sup>

• Centrifugation steps are on 150 g, low ramp, and brake.

• Centrifugation steps are 100 g, low ramp, and brake.

• CB starts proplatelet production earlier than adult sources. • Proplatelets can be harvested using Plts isolation protocols. • Proplatelets are easily activated, treat them as regular Plts.

• day 9–14: let the cells differentiate with half media change every 2–3 days.

• day 0 terminal differentiation: wash the cells twice with PBS and re-seed in medium supplemented with 10 U/ml EPO, 1 mg/ml holotransferrin, 2–5% plasma, 5 U/ml heparin at 2

Cellgro or Cell-Quin with 100 ng/ml FL, 50 ng/ml hSCF, 50 ng/ml TPO, 20 ng/ml IL-6 at 1 ×

 levels. • day 4: cells start to commit to the MK lineage (~10–20%), cells are collected and spun down

• day 0 terminal differentiation: re-seed cells in media (Cellgro or Cell-Quin) supplemented

• During terminal differentiation, the addition of tirofiban hydrochloride monohydrate

• day 11: cells will be committed to the MK lineage (80–100%) and consist of MKBLs and

• Cell collection from these days onward should be performed using 2 mL or larger pipettes and avoid the usage of hand pipettes to circumvent cell lysis and shear stress [157, 166].

• Flow cytometry techniques on unfixed MKs at this stage will induce granule release and

• 1 μM of SR-1 can be used to increase polyploidization and Plt production.

• day 12–16: MK cells will mature to late MKs and start producing proplatelets.

cells by MACS isolation from CB, BM, PBMC, or MPB and seed in

cells/ml.

with EPO, SCF, and DEX (concentration is same as day 0).

**Our culture protocol from CD34+ to MK**

cells/ml, 37°C, and 5% CO<sup>2</sup>

undisturbed as possible at stable CO<sup>2</sup>

at 200 g to start terminal differentiation.

and heparin is recommended.

proplatelet formation.

• Pipetting should be kept to minimal.

× 10<sup>6</sup> /ml.

264 Cell Culture

106

early MKs.

• day 0: collect CD34<sup>+</sup>

/ml medium supplemented

cells/ml, otherwise keep cells as

As described in this chapter, there are multiple protocols to culture RBCs and MKs from a variety of hematopoietic tissues. Depending on the goal (fundamental research, drug screening, or clinical applications), one should consider beforehand which source can be used. For clinical applications, the use of a fully defined unlimited source would be preferred. For this, iPSC (generated with nonintegrating method) hold great potential but differentiation toward RBCs and Plts has to be improved.
