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Thrombopoietin (TPO) is the primary cytokine regulator of megakaryopoiesis, exerting its effects through its specific cell- surface receptor, MPL. Mice deficient in either TPO or MPL produce approximately 10% of the normal levels of megakaryocytes and platelets under homeostatic conditions; furthermore, their platelet recovery is impaired under conditions of stress, such as bone marrow (BM) transplantation or exposure to cytotoxic drugs. MPL signaling also plays a critical role in maintaining the normal quantity and activity of hematopoietic stem cells (HSCs). In addition to stimulating megakaryopoiesis, TPO can support the survival and expansion of HSCs and megakaryocyte (MK) progenitors—either independently or in combination with other cytokines, including GM-CSF, IL-3, IL-6, IL-11, SCF, FLT ligand, FGF, and EPO.

TPO binds to its specific receptor protein, Mpl (TPOR), triggering the activation of Janus kinase 2 (Jak2) and the subsequent phosphorylation of the receptor. The primary signaling cascades activated by the TPO/Mpl interaction include the JAK/STAT, PI3K-Akt, and Ras-MAPK1/3 pathways.

The TPO Effector Reporter Cell model accurately simulates the in vivo TPO signaling transduction process; the underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the HEK293 Human TPO Effector Reporter Cell Model

Figure 2. Recombinant TPO Effector Reporter Cell constitutively expressing TPOR.

Figure 3. Dose Response of Ligands in TPO Effector Reporter Cell(C7).

Cell Resuscitation
1)Rapidly thaw the frozen cells in a 37 °C water bath for approximately 60 seconds. Once thawed (which may take slightly less or more than 60 seconds), immediately transfer the cell suspension from the cryovial into a 15 mL centrifuge tube containing 10 mL of pre-warmed  HEK293 Human TPO Effector Reporter Cell complete culture medium.
2)Centrifuge cells at 1000 rpm for 5 min to remove medium, then resuspend cells in 5 mL of pre-warmed complete medium.
3)Transfer the cell suspension into a T25 culture flask and incubate at 37 °C with 5% CO₂.
4)After approximately 24–36 hours, replace the medium or passage the cells to remove non-adherent dead cells.

Subculturing procedure
1)When the cell density reaches the appropriate confluency for passaging, wash the cells with PBS, then add 1 mL trypsin to detach the cells. When more than 80% of the cells detach upon gently tapping the culture flask, add complete culture medium to terminate digestion. Gently pipette to obtain a single-cell suspension, transfer to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.

2)Discard supernatant after centrifugation. Resuspend cells in fresh medium to a single-cell suspension and transfer to a new culture flask for continued growth.

Cell Freezing
After trypsinization and centrifugation of cells from each T75 flask or 10 cm culture dish, discard the supernatant. Add 2 mL of cryopreservation medium (90% FBS + 10% DMSO), gently resuspend thoroughly, and aliquot into two cryovials. Immediately place the cryovials into a controlled-rate freezing container (e.g., Nalgene 5100-0001), fill with isopropanol to the indicated level, and store at −80 °C. After 24 hours, transfer the cryovials to liquid nitrogen for long-term storage.