efficient rock pendulum feeder manufacturer in jordan

efficient rock pendulum feeder manufacturer in jordan

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Despite recent progress in the preparation of feeder cells for human induced pluripotent stem cells (hiPSCs), there remain issues which limit the acquisition of feeder cells in large scale. Approaches for obtaining feeder cells quickly on a large scale are in immediate need. To reach this goal, we established suspension-adhesion method (SAM) and three-dimensional (3D) suspension method (3DSM). In SAM, mouse embryonic fibroblast (MEF) growth were fully inhibited by 10 μg/ml mitomycin-C (MMC) in 0.5 hours, and the feeder cells generated display higher adherent and recovery rates as well as longer survival time compared to conventional method (CM). 3DSM, an optimized method of SAM in which MEFs were cultured and MMC treated in suspension, was developed to lower the costs and workload using CELLSPIN System. The yield of feeder cells is several times the yield of SAM while the adherent and recovery rates and the capacity of supporting hiPSCs growth were not sacrificed. Hence, 3DSM is an economical and easy way to generate large-scale feeder cells for hiPSCs cultures

Induced pluripotent stem cells (iPSCs) can be obtained from somatic cells by forced expression of a defined set of reprogramming factors, including either the combinations of Oct4, Klf4, Sox2, and c-Myc, or of Oct4, Sox2, Nanog, and Lin281,2,3,4. We previously reported to obtain iPSCs from human hair follicles-mesenchymal stem cells (hHF-MSC-derived iPSCs) using four Yamanaka factors (Oct4, Sox2, c-Myc and Klf4)5. These iPSCs are capable of self-renewal and differentiate into various cell types, feeder cells are required to support their growth while maintaining pluripotency

efficient feeder cells preparation system for large-scale

Feeder cells are known to produce growth factors, adhesion molecules, and extracellular matrix. The most widely used feedder cells include mouse embryonic fibroblasts (MEFs). Recently, a xeno-free cell culture method was established to avoid contamination by pathogens and animal proteins6,7. In that system, mouse feeder cells are replaced with human cells such as human fetal and adult fibroblasts8, human fetal muscle fibroblasts9, foreskin fibroblasts10, amniotic mesenchymal cells11, adipose-derived mesenchymal stem cells12, bone marrow mesenchymal stem cells13,14,15, placenta-derived mesenchymal stem cells16, multipotent mesenchymal stem cells of desquamated endometrium17, and decidua-derived mesenchymal cells18

In spite of recent progress in hiPSCs culture conditions, large-scale production of hiPSCs by robust and economical methods has been one of the major challenges for the translational realization of hiPSCs technology19. To achieve large-scale production of hiPSCs, a large-scale culture system for hiPSCs expansion using the E8 chemically defined and xeno-free medium has recently been developed20. However, the efficiency of human feeder layers in the maintenance of undifferentiated human embryonic stem cells (hESCs) growth is not as high as that of mouse feeder cells due to the lower level of secretion of activin A21. Although there are numerous chemically defined and xeno-free media such as mTeSR and StemPro conducive to the production of hiPSCs, the inclusion of human serum albumin and human sourced matrix proteins makes those conditions prohibitively expensive, impractical for routine use, and not truly completely defined, which limits their use in large-scale amplification of hiPSCs22,23. Thus, the feeder-based system remains an important method of hiPSCs propagation

Currently, feeder cells are mitotically inactivated either by gamma irradiation24,25,26,27,28,29,30 or MMC3,4,11,31,32,33,34. Gamma irradiation can treat more cells than MMC at one time, but the γ-ray radiation source of Cobalt-60 is rare and costly. The affordability, flexibility, and convenience of MMC make it a good routine protocol to prepare feeder cells. For the feeder-based culture system, MEFs of CF-1 strain mice characteristically exhibit active proliferation, high-density dependence, and being aging-prone at low-density, and are still the most common feeder source for hiPSCs cultures

In the conventional method (CM) for feeder cells preparation35, CF-1 MEFs of 80–90% confluence were inactivated and used as feeder cells to maintain hiPSCs or for the production of conditioned medium. However, low yield with high costs need to be optimized as individual dishes or flasks accommodate limited numbers of cells in CM. Failure to fully inactivate MEFs in stratified growth by MMC is another problem. At low density, however, MEFs are aging-prone and their supportive capacities for iPSCs are compromised. Hence, MMC processing time is inflexible. Therefore, it is necessary to find new approaches that not only can be used for the production of feeder cells on a large scale in a short time, but also can ensure that MEF proliferation is sufficiently inhibited. To this end, we recently established a suspension-adhesion method (SAM) and a three-dimensional (3D) suspension method (3DSM) by optimization of CM. These new methods for feeder preparation will promote the advances and applications of induced pluripotent stem cell technology

efficient feeder cells preparation system for large-scale

All methods were carried out in accordance with relevant guidelines and regulations of the Ethics Committee of the Norman Bethune College of Medicine, Jilin University. All experimental protocols were approved by the Ethics Committee of the Norman Bethune College of Medicine, Jilin University. Informed consent was obtained from all subjects. Animal experiments were performed in accordance with a protocol approved by Jilin University School of Medicine Animal Care and Use Committee [Animals use license: SYXK (Jilin) 2013-0005]. All mice were housed in a sterile environment and could access food and water commodiously as outlined in the institutional guidelines

CF-1 mouse embryonic fibroblasts (MEFs) were derived from day-12.5 embryo pools of CF-1 strain mice. The cells were cultured in MEF medium [DMEM supplemented with 10% FBS, 1% non-essential amino acids, 1 mM L-glutamine (all from Gibco, Invitrogen, USA)] and maintained at 100% confluence. hHF-MSC-derived iPSCs were cultured as described previously5. hHF-MSC-derived iPSCs were maintained on mitotically inactivated CF-1 MEFs in hESCs culture medium (80% DMEM/F12 supplemented with 20% KSR, 1% non-essential amino acids, 1 mM L-glutamine, 4 ng/ml human bFGF, 0.1 mM β-mercaptoethanol) (all from Invitrogen, USA). The hHF-MSC-derived iPSCs were split with 1 mg/ml collagenase type IV (Invitrogen, USA) for 30 min at 37 °C, at a ratio of 1:5 every 6–7 days. Conditioned medium was collected as described36. All manipulations and cultivations were performed in a good manufacturing practice (GMP)-compliant facility

CF-1 MEFs of passage 3 (P3) at 80–90% confluence were inactivated with 10 μg/ml of MMC (Hisun Pharmaceutical Company, China) for 0, 0.5, 1.0, 1.5, and 2.0 h at 37 °C. After the incubations, the cells were washed with PBS 6 times, trypsinized, centrifuged at 180 × g for 5 min, and re-suspended in MEF medium. Cells were counted and frozen for later use

efficient feeder cells preparation system for large-scale

We prepared feeder cells by SAM according to Fig. 1. Briefly, CF-1 MEFs of P3 were cultured for four days, digested to single cells with 0.25% trypsin/EDTA (Dalian Meilun Biotech Co., Ltd, China), and collected in 50 ml-centrifuge tubes. The cells were seeded at 8 × 104–1.1 × 105 cells/cm2 in 10 cm-dishes. MMC (10 μg/ml) was added after 2.0–3.0 h at 37 °C. Medium containing MMC was discarded 0.5–3.5 h post-treatment. The cells were then washed with PBS 6 times, trypsinized, centrifuged at 180 × g for 5 min, and resuspended in MEF medium. Cells were counted and frozen for later use

We prepared feeder cells by 3DSM according to Fig. 2. Briefly, CF-1 MEFs of P3 growing for four days were digested to single cells by 0.25% trypsin/EDTA, and collected into a 50 ml-centrifuge tube. Cells were transferred to spinner flasks with glass ball pendulum, which accommodate 25–1000 ml of volume, at a density of 0.5–1.3 × 106 cells/ml. MMC were added at 10 µg/ml. After incubation for 0.5, 1.0, 1.5, and 2.0 h at 37 °C, the cells were centrifuged at 180 × g for 5 min, washed with PBS 3 times, resuspended in MEF medium, counted, and cryopreserved for later use

The feeder cells of CM, SAM and 3DSM were plated at 1 × 105 cells per well in 24-well plates and counted using trypan blue staining solution(Dalian Meilun Biotech Co., Ltd, China) on days 1, 3, 5, and 7

efficient feeder cells preparation system for large-scale

The inhibitory effect of MMC on the proliferation of MEFs was measured using an EdU assay kit (Ribobio, Guangzhou, China) according to the manufacturer’s instructions. Briefly, inactivated MEFs were cultured in triplicate at 2 × 104 cells per well in 24-well plates. The cells were exposed to 50 μM EdU for 8–24 h at 37 °C. The cells were fixed with 4% formaldehyde for 15 min at room temperature and treated with 0.5% Triton X-100 for 20 min at room temperature for permeabilization. After 3× washes with PBS, the cells were treated with 100 μM 1 × ApolloR reaction cocktail for 30 min. Subsequently, cells were stained with 200 μl/well of Hoechst 33342 (5 μg/mL) for 30 min and visualized under a fluorescent microscope (Olympus, Japan). The number of EdU-positive cells was counted using open-source digitizing software (ImageJ 2.0.0, Wayne Rasband, National Institutes of Health, Bethesda, MD). The percentage of EdU-labeled cells was calculated as follows: EdU-positive cell number/Hochest-positive cell number

Inactivated MEFs freshly prepared with CM, SAM and 3DSM were plated at 1 × 106 cells/T25-flask. Non-adherent cells were collected and counted on the second day. Direct adhesion rates were then evaluated

The frozen MMC-inactivated MEFs of CM, SAM and 3DSM in T25-flasks were resuscitated on days 1, 7, 21, 28 respectively after cryopreservation and the non-adherent cells were collected and counted the following day to analysis the recovery rates of MMC-inactivated MEFs

efficient feeder cells preparation system for large-scale

Cell cycle assay was determined by flow cytometry (BD, USA). Briefly, 1 × 106 inactivated and cryopreserved MEFs of CM, SAM, and 3DSM were plated in T25 flasks. The cells were harvested and fixed in 70% ice-cold ethanol for 24 h, then stained with propidium iodide (PI). The different cell cycle phases were analyzed using a FACS Calibur instrument

High performance liquid chromatography-Mass Spectrometry/Mass Spectrometry (HPLC-MS/MS) was performed as described37 with modifications. Briefly, a set of MMC calibration solutions with different concentrations were made from a standard stock solution of 1 mg/ml MMC by dilution with methanol: water (1:1, v/v) solution. Aliquots (400–500 μl) were analyzed by HPLC-MS/MS to construct a standard curve. All chomatographic experiments were carried out at room temperature using HPLC (Agilent technologies, USA) equipment. MS/MS analysis was performed using LCQ Deca XP plus equipment (Agilent technologies, USA) with an ESI source in the positive ion mode. The daughter ions with m/z 274 for MMC were monitored via an ion trap mass analyzer

hHF-MSC-derived iPSCs were harvested by collagenase type IV. After settling, the supernatant was aspirated and the MEF medium was replaced to remove the MEF. hHF-MSC-derived iPSCs were transferred to petri dishes in the MEF medium. After an 8 days floating culture, embryoid bodies were transferred to gelatin-coated plates and were then incubated for another 16 days. After the incubation, the cells were fixed with 4% paraformaldehyde in PBS and then incubated in PBS containing 5% normal goat serum (Maixin Biotech, Fuzhou, China), 1% bovine serum albumin (BSA, Biotopped, China), and 0.2% Triton X-100. The primary antibodies were as follows: anti-alpha smooth muscle actin (α-SMA, R&D, USA), anti-alpha fetoprotein polyclonal antibody (AFP, R&D, USA), and anti-Nestin (R&D, USA). vimentin(Cell Signaling, USA), desmin (Cell Signaling, USA), βIII-tubulin (Cell Signaling, USA). The secondary antibodies were Alexa 555-labeled anti-mouse IgG (1:1000, Cell Signaling, USA). Nuclei were stained with 1 mg/ml Hoechst 33342 (Invitrogen, USA)

efficient feeder cells preparation system for large-scale

The hHF-MSC-derived iPSCs were injected intramuscularly into non-obese diabetic/severe combined immune deficient (NOD/SCID) mice in DMEM containing 10% FBS (3 × 106 cells per site). After 12 weeks, teratomas were retrieved from the injection site, dissected, and fixed with 10% formaldehyde in PBS. Paraffin embedded tissue sections were then prepared and analyzed with Hematoxylin and Eosin (H&E) staining

We prepared feeder cells by conventional method (CM) as previous reports3,4,35. Briefly, we harvested MEFs from embryonic day 12.5 embryo pools of CF-1 mice. The primary cells contained a variety of different types of cells. CF-1 MEFs became more homogeneous at passage 3 (P3) and were used as feeders. MEFs were treated with 10 μg/ml MMC for 0, 0.5, 1.0, 1.5, or 2.0 h respectively. Then, the culture media were replaced with MEF media and cell numbers were counted on days 1, 3, 5, and 7. As shown in Fig. 3, phase contrast microscopy images and cell counts demonstrated that the number of cells significantly increased (P < 0.01) in the culture on days 1, 3, 5, and 7 after being treated with 10 µg/ml MMC for 0.5 h by CM, indicating that MMC treatment for 0.5 h failed to inhibit MEF proliferation in CM. However, the number of cells significantly decreased in CM when using MMC for 1.0, 1.5, and 2.0 h, and statistically significant differences were found between days 1, 3, 5, and 7 (P < 0.01). It is worth mentioning that some of the cells were unhealthy in the MMC 1.0–2.0 h groups and died in three days

Inhibition of MEF growth by MMC in CM. (A) Phase contrast microscopy images of MEFs treated with 10 µg/ml MMC for 0–2.0 h in CM and observed on days 1, 3, 5 and 7. Bars = 40 μm. (B) Cell counts of inactivated MEFs in each well of 24-well plate (Student’s t test and ANOVA; **P < 0.01 versus each group of days 1, 3, 5 and 7)

efficient feeder cells preparation system for large-scale

To optimize CM, we first prepared feeder cells according to Fig. 1. Several reports suggested that 10 μg/ml MMC is sufficient to inhibit proliferation of MEFs21,36,38,39,40,41,42,43,44,45. To identify the optimal time of inhibition in suspension-adhesion method (SAM), we performed a time course experiment, exposing the MEFs to 10 µg/ml MMC for 0–6.0 h. Phase contrast microscopy images and cell counts (Fig. 4A,B and Fig. S1) showed that MEFs statistically significant increased in the cell numbers on days 1, 3, 5, and 7 without MMC treatment (P < 0.01). MEFs exposed to MMC for 0.5–4.0 h did not change in number over 7 days. The number of cells did not perceptibly increase or decrease on days 1, 3, 5, or 7 for cells treated with MMC for 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, or 4.0 h, and no statistically significant differences were found in cell numbers on days 1, 3, 5, or 7. However, the number of cells decreased on days 1, 3, 5, and 7 for MEFs treated with MMC for 4.5, 5.0, and 6.0 h (P < 0.05) probably due to cell death. Take together, our data suggest that treatment with 10 µg/ml MMC for 0.5–4.0 h is sufficient to suppress the proliferation of MEFs but maintains acceptable viability of feeder cells. Thus, we prepared feeder cells with SAM using 10 µg/ml MMC for 0.5–4.0 h

Inhibition of MEF growth by MMC in SAM. (A) Phase contrast microscopy images of MEFs treated with 10 µg/ml MMC for 0–2.0 h in SAM and observed on days 1, 3, 5 and 7. Bars = 40 μm. (B) Cell counts of inactivated MEFs in each well of 24-well plate (Student’s t test and ANOVA; **P < 0.01 versus each group of days 1, 3, 5 and 7). (C) EdU-positive cells and representative images of fields show red-colored proliferation cells. Hoechst 33342 staining was performed to detect nuclear localization. Bars = 40 μm. (D) The number of EdU-positive cells were counted using ImageJ (Student’s t test; ***P < 0.001)

To further validate the suppressive effect of MMC on the proliferation of MEFs, we labeled the cells with EdU and determined the cell proliferation rate after MMC treatment (Fig. 4C,D and Table S1). There were statistically significant differences between untreated cells and those treated with MMC for each time point between 0.5–3.5 h and gamma ray (P < 0.001). However, there was no significant difference between the gamma ray group and cells treated with MMC for 0.5–3.5 h (data of 2.0–3.5 h are not shown). Together, these results further confirmed that 10 μg/ml MMC for 0.5–3.5 h was sufficient to inhibit MEF proliferation

efficient feeder cells preparation system for large-scale

Both the SAM and CM require MEFs to attach to the flasks/dishes, which limits the processing efficiency of the feeder cells. To increase the processing efficiency and reduce costs (Table 1), we used the three-dimensional (3D) suspension method (3DSM) to re-optimize the SAM for the preparation of feeder cells (Fig. 2). MEFs were treated with 10 μg/ml MMC for 0–2.0 h. Then, we observed and counted cell numbers on days 1, 3, 5, 7. Phase contrast microscopy images and cell counts (Fig. 5A,B) demonstrated that without MMC the number of MEFs significantly increased on days 1, 3, 5, and 7 in 3DSM (P < 0.01). The number of cells did not change on days 1, 3, 5, or 7 after treatment with MMC for 0.5–2.0 h

Inhibition of MEF growth by MMC in 3DSM. (A) Phase contrast microscopy images of MEFs treated with 10 µg/ml of MMC for 0–2.0 h in 3DSM and observed on days 1, 3, 5 and 7. Bars = 40 μm. (B) Cell counts of inactivated MEFs in each well of 24-well plate (Student’s t test and ANOVA; **P < 0.01 versus each group of days 1, 3, 5 and 7)

To determine the quality of feeder cells prepared by SAM and 3DSM, we compared the shortest processing time (SPT) of inhibition MEF proliferation, the direct adherent rates (DAR), recovery rates (RR) and survival time (ST) of MEFs harvested from SAM, 3DSM and CM

efficient feeder cells preparation system for large-scale

The aforementioned data showed that MMC for 0.5 h was sufficient to inhibit the proliferation of MEFs in SAM and 3DSM (Figs 4A,B and 5A,B). But MMC for 0.5 h failed to inhibit MEF proliferation in CM (Fig. 3A, B). Thus, SAM and 3DSM were more efficient than CM in terms of the suppression of MEF proliferation. In addition, the number of MEFs in SAM and 3DSM did not perceptibly increase or decrease on days 1, 3, 5, or 7 for those treated with MMC for 0.5–2.0 h. But some of the MEFs treated with MMC for 1.0–2.0 h in CM were unhealthy and died in three days. Thus, it seems that the feeder cells prepared in SAM and 3DSM may be higher quality as those prepared in CM

Next, we compared the DAR of inactivated MEFs treated with MMC for 0.5, 1.0, 1.5, and 2.0 h in CM with SAM and 3DSM (Fig. 6A). The DAR of inactivated MEFs treated with MMC for 0.5–2.0 h in SAM could reach 96% ± 2% and 3DSM could also reach 93% ± 4%, however, the rate in CM was 82% ± 5%. Our data indicated that the DAR of MEFs in SAM and 3DSM were higher than that in CM

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