**Our culture protocol from human PBMC to RBC**

	- Optional: to remove remaining RBCs after Ficoll purification, the use of RBC lysis buffer is suggested.
	- Optional: to remove remaining lymphocytes, purify the culture by density centrifugation on a 1.075 g/ml Percoll gradient.

• day 6–20: put the cells daily or every second day to 0.5–0.7 × 10<sup>6</sup> /ml medium supplemented with EPO, SCF, and DEX (concentration is same as day 0).

**3. Conclusions**

RBCs and Plts has to be improved.

**Acknowledgements**

**Conflict of interest**

**Abbreviations**

AGM aorta-gonad-mesonephros AhR aryl hydrocarbon receptor

bFGF basic fibroblast growth factor

BMP4 bone morphogenetic protein 4

cRBC cultured red blood cell

BM bone marrow

CB cord blood

DEX dexamethasone

dox doxycycline

None.

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

Erythropoiesis and Megakaryopoiesis in a Dish http://dx.doi.org/10.5772/intechopen.80638 265

We would like to thank Esther Heideveld, Joan Gallego Murillo, Department of Hematopoiesis and Laboratory for Cell Therapy (Sanquin, Amsterdam) for their experimental and scientific input. We are grateful for the support by the Ministry of Health (PPOC: 11-035, 15-2089), the Landsteiner Foundation for Blood Transfusion Research (LSBR1141), the European Union (FA H2020-MSCA ITN-2015, RELEVANCE), and the Netherlands Organization for Scientific

Research (NWO/ZonMw 40-41400-98-1327; 40-00812-98-12128).


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

	- Re-seed cells when concentration exceeds 2.5 × 10<sup>6</sup> cells/ml, otherwise keep cells as undisturbed as possible at stable CO<sup>2</sup> levels.
	- During terminal differentiation, the addition of tirofiban hydrochloride monohydrate and heparin is recommended.
	- Pipetting should be kept to minimal.
	- 1 μM of SR-1 can be used to increase polyploidization and Plt production.
	- 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].
	- Centrifugation steps are on 150 g, low ramp, and brake.
	- Flow cytometry techniques on unfixed MKs at this stage will induce granule release and proplatelet formation.
	- 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.
