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Is there any alternative to cryopreserve cell lines without -80 freezer?

Is there any alternative to cryopreserve cell lines without -80 freezer?


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My university's -80 freezer just broke down and the repair will take 4 months according to the vendor. Is there any other way to preserve cells? Maintaining lot of flasks until the freezer is repaired is not viable option.

Is there any alternative to cryopreserve cell lines without -80 freezer?.


A tank of liquid nitrogen is not just an alternative, but a much better substitute, IMHO. The problem of gradually cooling inside a isopropanol container can be overcome by using a box full of dry ice (-79°C as I recall).


Cryopreservation Freezing Methods and Equipment

The objective of cryopreservation is to minimize damage to biological materials, including tissues, mammalian cells, bacteria, fungi, plant cells, and viruses, during low temperature freezing and storage. When performed correctly and accounting for cell and tissue specific criteria, cryopreservation can provide a continuous source of tissues and genetically stable living cells for a variety of purposes, including research and biomedical processes. This article will describe the workflow, methods and equipment used for freezing, storage and thawing samples.


Bambanker Serum Free Cell Freezing Medium, Bulldog Bio

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2. Cryopreservation

2.1. Cryopreservation procedure

Cryopreservation is the use of very low temperatures to preserve structurally intact living cells and tissues for a long period of time.2 Depending on the cell types or given cells among different mammalian species, there is great diversity in cryobiological response and cryosurvival during the freezing and thawing cycle ( Fig. 1 and Table 1 ).5 Cryopreservation processes can generally be grouped into the following types: (1) slow freezing8, 9 (2) vitrification, which involves the solidification of the aqueous milieu of the cell or tissue into a noncrystalline glassy phase10 (3) subzero nonfreezing storage and (4) preservation in the dry state.11 Generally, the storage of mammalian cells in the dry state is not readily possible because of difficulties in introducing the disaccharide trehalose (disaccharide of glucose, 342 Da)12 and amino acids (used as preservatives in plants) into the intracellular region.13 The major steps in cryopreservation are (1): the mixing of CPAs with cells or tissues before cooling (2) cooling of the cells or tissues to a low temperature and its storage (3) warming of the cells or tissues and (4) removal of CPAs from the cells or tissues after thawing.14 The appropriate use of CPAs is therefore important to improve the viability of the sample to be cryopreserved.

2.2. Cryoinjury

The exact mechanism of cryoinjury, which is the damage of cells associated with the phase changes of water in both extra- and intracellular environments at low temperatures, has not been clearly established.5 The cooling and thawing velocities can largely affect the physicochemical and biophysical reactions, altering the survival rate. Cryoinjury mechanisms involving osmotic rupture caused by extra- or intracellularly concentrated solutes and intracellular ice formation have been highly suggested,15, 16, 17 both processes of which are dependent on the cooling rate ( Fig. 1 ).5, 18 In addition, cell viability limits are defined primarily in terms of an intact plasma membrane that retains normal semipermeable properties. Indeed, conditions that allow the plasma membrane to survive may not allow the survival of critical organelles within cells.5

2.3. CPAs

The CPA, which is usually a fluid, reduces the freezing injury from the cryopreservation process ( Fig. 1 ). CPAs should be biologically acceptable, be able to penetrate the cells, and have low toxicity.2 Various CPAs have been developed ( Table 2 ) and are used to reduce the amount of ice formed at any given temperature, depending on the cell type, cooling rate, and warming rate.2 In order to achieve the best survival rate of cells and tissues, the sample volume, cooling rate, warming rate, and CPA concentrations should be optimized depending on the different cell types and context of tissues.18 It should be mentioned that the macroscopic physical dimension of the tissue is a major point to be defined in a cryopreservation protocol because of heat and mass transfer limitations in these bulk systems.1, 18 CPAs can be divided into two categories: (1) cell membrane-permeating cryoprotectants, such as dimethyl sulfoxide (DMSO), glycerol,19 and 1,2-propanediol and (2) nonmembrane-permeating cryoprotectants, such as 2-methyl-2,4-pentanediol and polymers such as polyvinyl pyrrolidone, hydroxyethyl starch, and various sugars.1, 4 Unlike synthetic chemicals, biomaterials such as alginates, polyvinyl alcohol, and chitosan can be used to impede ice crystal growth, along with traditional small molecules.4 The direct inhibition of ice crystal formation and application of antioxidants and other compounds have been used to attempt to reduce cell death from processes such as apoptosis during the freezing and thawing cycle.20, 21, 22, 23 Common CPAs are briefly addressed in the following subsections and Table 2 .

Table 2

Commonly used cryoprotective agents and their uses

Cryoprotective agentsMembrane permeabilityPossible toxicity30Applied in cryopreservation
Cell Banker seriesYesUnknown but less than that of DMSOAdipose tissue-derived stem cells29
Amniotic fluid
Bone marrow36
Mammalian cells
Synovium36
Dimethylsulfoxide
(DMSO)
YesReduction in heart rate
Toxicity to cell membrane
Adipocyte tissue36
Amniotic fluid and umbilical cord36
Bone marrow36
Dental pulp36
Embryo (combined with EG or propylene glycol)44
Embryonic stem cells (alone or combined with EG)37
Hepatocytes11
Microorganisms26
Oocyte (combined with EG)37, 45
Platelet27
Teeth36
Testicular cell/tissue
Ethylene glycol (EG)YesGastrointestinal irritation
Pulmonary edema
Lung inflammation
Amniotic fluid36
Dental pulp36
GlycerolYesRenal failureAmniotic fluid
Microorganisms26
Red blood cell37, 38, 39Spermatozoa
Teeth36
TrehaloseNoRelatively less toxicAdipose-derived stem cells (combined with vitrification)28
Embryo (combined with vitrification)
Ovarian tissue (combined with vitrification)
Red blood cell38
Spermatozoa12
Stem cells (combined with propylene glycol)37
Propylene glycol (1,2-propanediol)YesImpairment in the developmental potential of mouse zygotesEmbryo30, 31
Hepatocytes11

2.3.1. Glycerol

Polge et al24 discovered the cryoprotective effect of glycerol in 1949, and this polyol compound remained the most effective of additives until the protective effect of DMSO was demonstrated by Lovelock and Bishop in 1959.25 Glycerol is a nonelectrolyte and thus may act by reducing the electrolyte concentration in the residual unfrozen solution in and around a cell at any given temperature. It is widely used in the storage of bacteria and animal sperm.26

2.3.2. DMSO

First synthesized by the Russian scientist Alexander Zaytsev in 1866, DMSO has been commonly used for the cryopreservation of cultured mammalian cells because of its low cost and relatively low level of cytotoxicity.25, 27 Like glycerol, DMSO acts by reducing the electrolyte concentration in the residual unfrozen solution in and around a cell at any given temperature. However, a decline in the survival rate and the induction of cell differentiation caused by DNA methylation and histone alteration have been reported.28, 29 These negative effects of DMSO in cryopreservation create some difficulties for its use in routine clinical applications.

2.3.3. Polymers

The entrapment of CPAs within a capsule during cell resuspension in an encapsulating material is another strategy for the modulation of cell location.4 Among encapsulating materials, synthetic nonpenetrating polymers can provide cryoprotection of cells within the scaffold, thereby bypassing the limitations of diffusion in higher-dimensional cryopreservation.4 Vinyl-derived polymers, such as polyethylene glycol (C2nH4n+2On+1, molecular weight: 200� Da), polyvinyl alcohol [(C2H4O)n, molecular weight: 30� kDa), and hydroxyethyl starch (130� kDa), have a capacity to decrease the size of formed ice crystals.4, 30, 31

2.3.4. Proteins

Sericin is a water-soluble sticky protein (� kDa) isolated from the silkworm cocoon and has been developed as a fetal bovine serum- or DMSO-replacing CPA for human adipose tissue-derived stem or progenitor cells, or hepatocytes.26, 27 Small antifreeze proteins derived from marine teleosts or fishes have also attracted attention as CPAs.32

2.3.5. Cell Banker series

A newly developed Cell Banker series (Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan) allows for rapid cell cryopreservation at � ଌ, and has been shown to achieve better survival rates following freezing and thawing.29, 33 The Cell Banker series of cryopreservation media contain 10% DMSO, glucose, a prescribed high polymer, and pH adjustors.33 Serum-containing Cell Bankers 1 and 1+ can be used for the cryopreservation of almost all mammalian cells. Indeed, conventional cryopreservation media include fetal bovine serum, which contains a mixture of growth factors, cytokines, and undefined substances such as bovine exosomes, rendering its use forbidden in the establishment of a standardized cryopreservation protocol for clinical use in humans.34 In this aspect, the nonserum-type Cell Banker 2 is optimal for the cryopreservation of cells in serum-free culture conditions. Cell Banker 3 (or Stem cell Banker) is composed of 10% DMSO and other inorganic compounds (US20130198876) and satisfies the criterion of a chemically defined known ingredient that is xeno free and is thus suitable for the preservation of somatic stem cells and induced pluripotent stem cells.


CHAPTER 4 - Cryopreservation of Human Embryonic Stem Cells

Cryopreservation is used to stabilize cultures at specific points in time with specific genetic characteristics. Without the ability to cryo-preserve cell lines, scientists are forced to subculture them continuously, during which time the cells may accumulate genetic changes, and become heterogeneous. Cryopreservation allows in producing a bank of stock vials at specific passages with specific genetic characteristics. Using validated stock vials to initiate new experiments maximizes the long-term usefulness of a cell line, and minimizes experimental variation. During cryopreservation, most of the water is removed from the interior of cells, and is converted to ice. It stops metabolism, and allows cells to be stored at low temperatures for long periods of time. However, recovery of hESCs from cryopreservation is sometimes very poor. Because of the slow growth rate of hESCs, the time from thawing of the vial to the formation of the cultures suitable for experimentation can be weeks to months.


Results

Two clinical-grade hESC lines, MasterShef7 (MShef7) and RC17, were chosen for this study since they efficiently differentiated into mDA neural progenitor cells and into mDA neurons using a modified 2D floor plate protocol (Kriks et al., 2011 Kirkeby et al., 2017). Briefly, hESCs were plated onto a matrix of Laminin-111 in the presence of dual-Smad inhibition (SB431542 + LDN193189), 0.9𠄱 μM GSK3β inhibitor (CHIR99021), and 600 ng/ml Sonic hedgehog (SHH-C24II). FGF8b and heparin were added at day 9 of differentiation, and brain derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF) were added at day 11 (Figure 1A). The differentiating mDA neural progenitor cells can be conveniently cryopreserved at day 11 or day 16 of differentiation, since these are time-points when the cells are lifted and re-plated in the protocol. However, for all experiments in this study, cells were frozen at day 16 since cells at this stage of maturity can be transplanted into pre-clinical rat models of Parkinson’s (Kirkeby et al., 2012), and no further re-plating steps are required to produce mature mDA neurons. In order to examine the in vitro differentiation potential of the cryopreserved cells, the mDA neural progenitor cells are thawed onto Laminin-111 in the presence of neurotrophic and neuronal maturation factors to resume differentiation into mDA neurons up to day 45 (Figure 1A). At the point of freezing (day 16), the vast majority of cells expressed the mDA markers, LMX1A, FOXA2, and Engrailed1 (EN1) as determined by immunocytochemistry (Figures 1B,C), and transcripts for these three transcription factors increased significantly during the differentiation process (Figure 1D). Flow cytometry for CORIN, a ventral floor plate marker, demonstrated that MShef7 and RC17 hESCs routinely produced �% CORIN-positive cells by day 16 of differentiation (Figures 1E,F). Furthermore, immunocytochemistry at day 16 revealed that LMX1A-positive cells exhibited cytoplasmic or membrane immunostaining for CORIN (Figure 1G).

Figure 1. Differentiation of human embryonic stem cells (hESCs) into midbrain dopaminergic (mDA) precursors for cryopreservation. (A) Schematic of the mDA differentiation protocol. hESCs are plated as clumps on day 0 in the presence of dual SMAD inhibitors, SB431542 + LDN193189, Sonic hedgehog (SHH), and the GSK3β inhibitor, CHIR99021. At day 9, the medium is switched to contain only FGF8b and the co-factor heparin, and cells are lifted and re-plated at day 11 when the neurotrophic factors BDNF and GDNF are added. At day 16, the cells are lifted and counted for cryopreservation. Live/dead cell counts were performed immediately upon thawing and at 24 h after thawing (day 17). mDA neuronal maturation was conducted in the presence of BDNF, GDNF, db-cAMP, ascorbic acid (AA), and the Notch inhibitor DAPT, up to day 45. Co-immunostaining for midbrain floor plate markers, FOXA2 with LMX1A (B), and FOXA2 with EN1 (C), at day 16 revealed near homogenous expression at the point of cryopreservation. Scale bar, 120 μm. (D) RT-qPCR analysis of LMX1A, FOXA2, and EN1 expression relative to TBP at Day 0, 11, and 16 of differentiation for three independent experiments (n = 3). (E) FACS analysis for CORIN expression at day 16 showed the majority of cells expressed this ventral floor plate marker. The control FACS with rat isotype control antibody is shown in the inset. (F) Percentage CORIN-positive cells as determined by FACS for undifferentiated day 0 MShef7 hESCs (n = 2) and RC17 hESCs (n = 2), and day 16 MShef7-derived (n = 8) and RC17-derived (n = 6) mDA neural progenitor cells. (G) Co-immunostaining of day 16 MShef7-derived mDA neural progenitor cells for CORIN and LMX1A. Scale bar, 120 μm.

Human embryonic stem cell-derived mDA neural progenitor cells were cryopreserved at a density of 𢏁 × 10 7 cells/ml in a total volume of 100 μl in six (6) different clinical-grade cryopreservation media that are commercially available (Table 1). This relatively small volume was used to facilitate uniform thawing and the high cell density was published to improve cell viability when compared to cells frozen at 𢏁 × 10 6 cells/ml (Terry et al., 2010). The media investigated included (i) STEM-CELLBANKER ® (SCB), (ii) Synth-a-Freeze TM Medium (SYF), (iii) PSC Cryopreservation Medium (PSC), (iv) CryoStor ® CS10 Freeze Medium, (v) CryoStor ® CS5 Freeze Medium, and (vi) Cellvation Cryopreservation Medium (CV). CS5 contained 5% DMSO (v/v), while CV is a DMSO-free cryopreservation medium. The other four cryopreservation media contained 10% DMSO (v/v). HypoThermosol ® , a 4ଌ DMSO-free hibernation medium, was used as a negative control for these experiments. Cooling rates were varied from 0.5 to 2ଌ/min using a VIA Freeze TM Duo controlled-rate freezer starting from 4ଌ and stopping at �ଌ before transferring vials to vapor phase liquid nitrogen storage (Figure 2). The vapor phase of liquid nitrogen (below �ଌ) was chosen since this is below the glass transition temperature of cells in cryoprotectant (∼�ଌ), and biological material is known to be highly stable at this temperature with no changes in cell viability for over 1 year (Silani et al., 1988 Massie et al., 2013 Meneghel et al., 2019). The recovery of mDA neural progenitor cells was performed by either thawing vials slowly in air at 4ଌ or rapidly in a water-bath set to 37ଌ. Cell viability was assessed by Trypan blue exclusion immediately upon thawing prior to plating in mDA neuronal differentiation conditions, and again at 24 h post-thawing. The latter cell viability test was performed since cryopreservation-induced apoptosis is reported to occur in some cell types 12� h after thawing (Baust et al., 2001). In order to gain a more detailed account of the cell population, both floating cells and attached cells were counted at 24 h post-thawing (Figure 2). However, even this will not capture the fate of all cells, since some cryopreserved cells will be mechanically destroyed during the freezing and thawing process due to irreversible intracellular ice crystal formation (Murray and Gibson, 2020). These destroyed cells will be missing from the live/dead and attached/floating cell counts. For this reason, we calculate both cell viability and cell recovery, the latter defined as a percentage of cells initially frozen.

Figure 2. Cryopreservation experimental procedure. (A) At day 16 of mDA differentiation, cells were lifted, counted, and resuspended in a clinical-grade cryopreservation medium prior to transferring to FluidX cryovials. (B) Cryovials were placed in the VIA Freeze controlled-rate freezer and cells were cryopreserved at a constant rate of cooling between 0.5 and 2ଌ/min to a final temperature of �ଌ. (C) Cryovials were transferred to the vapor phase of liquid nitrogen for long term storage. (D) Cryopreserved cells were thawed at 4 or 37ଌ. (E) A live/dead cell count was performed immediately after thawing (0 h count) prior to plating the cells. (F) 24 h after plating cells (day 17 of differentiation) the non-adherent floating cells and the adherent attached cells were subjected to live/dead cell counts (� h𠅏loating” count and � h𠅊ttached” count, respectively).

Rho-associated kinase (ROCK) inhibitors improved viability of single hESCs upon passaging and after cryopreservation (Watanabe et al., 2007), and they significantly improved the recovery of cryopreserved hESC-derived cardiomyocytes (Kim et al., 2011). Therefore, we first compared Y27632 (10 μM), a cell-permeable ROCK inhibitor, with the commercial formulation, RevitaCell TM , which contains a ROCK inhibitor and antioxidants. At 24 h post-thawing both RevitaCell TM and Y27632 significantly increased the number of attached live mDA neural progenitor cells (Figure 3A), and significantly reduced the number of floating cells (Figure 3B). There were no significant differences between RevitaCell TM and Y27632 by these measures. Since RevitaCell TM is manufactured at clinical-grade for medical devices (21 CFR Part 820 and ISO 13485), it was used for all subsequent experiments.

Figure 3. Assessment of cell viability after cryopreservation in the presence or absence of ROCK inhibitors, and using seven different clinical-grade media. (A,B) Cell viability of mDA cells after cryopreservation in the absence of ROCK inhibitors or in the presence of RevitaCell TM or Y27632. (A) Live/dead attached cell numbers at 24 h after thawing, and (B) total number of floating cells at 24 h (n = 2 experimental replicates). (C) Comparison of cell viability after cryopreservation in PSC Cryopreservation Medium (PSC), CryoStor ® CS10 Freeze Media (CS10), CryoStor ® CS5 Freeze Media (CS5), STEM-CELLBANKER ® (SCB), Synth-a-Freeze Medium (SYF), Cellvation Cryopreservation Medium (CV), and Hypothermosol ® (Hypo) immediately after thawing. At 24 h post-thawing live/dead cell counts of the attached cells (D) and total floating cells (E) were determined (n = 3 experimental replicates), and compared to freshly passaged cells (n = 6 experimental replicates). Live cells as a percentage of initial cell number frozen immediately upon thawing (F), and attached cells at 24 h post-thawing (G) (n = 3 experimental replicates). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 one-way ANOVA with Tukey’s multiple comparisons test.

We conducted a side-by-side comparison of six commercially-available, clinical-grade cryopreservation media, and a DMSO-free hibernation medium, HypoThermasol ® (Hypo) (Table 1). Cell viability immediately upon thawing was not significantly different for all cryopreservation media, nor was it significantly different to the hibernation medium (Figure 3C). However, at 24 h post-thawing, there were significant differences in cell viability for the different cryopreservation media tested. Cells frozen in the hibernation medium, Hypo, gave the lowest level of cell viability, while PSC Cryopreservation Medium was the only one that was not significantly different from freshly passaged cells (Figure 3D). Cellvation, a DMSO-free cryopreservation medium, was not significantly worse than the DMSO-containing cryopreservation media, while Hypo was significantly worse than PSC, CS10, CS5, and SCB cryopreservation media (Supplementary Table S2). The number of floating cells at 24 h post-thawing was significantly higher for cells frozen in SYF, CV, and Hypo media, but not for cells cryopreserved in PSC, CS10, CS5, or SCB media (Figure 3E). When the data are analyzed for cell recovery, defined as a percentage of viable cells to the initial number of frozen cells, there were no significance differences in cell recovery immediately upon thawing for any of the media (Figure 3F), but at 24 h post-thawing the DMSO-free media, CV and Hypo, had significantly lower percentages of live recovered cells than the best-performing medium, PSC (Figure 3G). While the performance of CV and Hypo were not significantly different from each other by this measure, Hypo was significantly poorer than all the DMSO-containing cryopreservation media, while the DMSO-free CV media was not statistically different from SCB and SYF media, which contain 10% DMSO (v/v) (Supplementary Table S2).

Since PSC was the best-performing cryopreservation medium in the head-to-head comparison (Figure 3D), we attempted to further improve cell recovery by investigating three different cooling rates and two thawing conditions. We also included CS5 and CV media, which contained 5% DMSO and 0% DMSO, respectively, since the presence of DMSO could affect downstream differentiation (Pal et al., 2012). For PSC media, 1 and 2ଌ/min were significantly better than 0.5ଌ/min at 24 h post-thawing, while there were no significant differences for cells frozen in CS5 medium (Figures 4A,B). For the DMSO-free CV medium, the fastest freezing rate of 2ଌ/min was significantly better than the slowest rate of 0.5ଌ/min (Figure 4C). Although it is common practice to thaw cells rapidly in a warm water-bath, we found that slow thawing in cool conditions (10 min at 4ଌ) was significantly better than fast thawing at 37ଌ for cells frozen in PSC media (Figure 4D). However, mDA neural progenitor cryopreserved in CS5 or CV media did not show significant differences between the slow and rapid thawing conditions (Figures 4E,F). mDA neural progenitor cells differentiated from another hESC line, RC17, also exhibited excellent survival 24 h post-thawing after cryopreservation in PSC medium (Figure 4G). In agreement with the MShef7 data, slow thawing at 4ଌ was equivalent, if not superior, to rapid thawing at 37ଌ for RC17-derived mDA neural progenitor cells frozen in PSC medium (Figure 4G). Cells frozen in the 5% DMSO medium, CS5, did not recover as well as cells cryopreserved in PSC medium however, there was a trend toward better survival in the rapid 37ଌ thawing condition for mDA neural progenitor cells differentiated from both MShef7 and RC17 cell lines (Figures 4E,H).

Figure 4. Cell recovery at different cooling rates and thawing conditions. Percentage of live attached cells recovered 24 h after thawing for cells cryopreserved at different cooling rates, 0.5, 1.0, and 2.0ଌ per minute, in PSC (A), CS5 (B), or CV (C) cryopreservation media in three independent experiments (n = 3). *p < 0.05, **p < 0.01, one-way ANOVA with Tukey’s multiple comparisons test. (D𠄿) Cell recovery at 24 h post-thawing for mDA cells frozen in PSC (n = 5), CS5 (n = 3), or CV (n = 3) cryopreservation media and thawed at 4 or 37ଌ in three to five independent experiments. *p < 0.05, unpaired t-test with Welch’s correction. (G,H) Cell recovery of RC17-derived mDA cells at different thaw rates (37 versus 4ଌ) in PSC and CS5 cryopreservation medium (n = 2 experimental replicates).

Next we directly compared the differentiation potential of MShef7 and RC17-derived non-frozen cells to mDA neural progenitor cells frozen at day 16 in the optimized condition: PSC cryopreservation medium cooled at 1ଌ/min and thawed slowly at 4ଌ. At 45 days of DA neuronal differentiation, there were no gross differences in the production of neurons for cells frozen at day 16 when compared to non-frozen cells in terms of morphology or expression of the pan-neuronal marker βIII-tubulin and the DA marker, tyrosine hydroxylase (TH) (Figures 5A,B). Quantification of TH immunostaining with respect to βIII-tubulin expression for both MShef7 and RC17 hESC-derived neurons at day 45 of differentiation did not reveal any significant differences between non-frozen cells and cells frozen at day 16 of the protocol (Figure 5C). Gene expression analysis of TH, and mDA transcription factor markers, NURR1 and PITX3, also did not reveal any significant differences between frozen and non-frozen mDA neural progenitor cells differentiated from both MShef7 and RC17 hESC lines (Figure 5D).

Figure 5. Marker analysis of continuously differentiated mDA neurons versus neurons that were cryopreserved at day 16 as mDA neural progenitor cells. Immunofluorescence staining for the pan-neuronal marker, βIII-tubulin (βIII), and dopaminergic marker, tyrosine hydroxylase (TH), at day 45 of differentiation of MShef7 hESCs without freezing (A) or with freezing (B) at day 16. Zoomed-in areas designated by dashed lines. Scale bars, 220 μm. (C) Image quantification of TH immunostaining as a fraction of βIII-tubulin immunostaining for non-frozen and frozen mDA neurons differentiated from MShef7 and RC17 hESC lines (n = 1 experimental replicate for each cell line, three to four images per condition). (D) RT-qPCR gene expression analysis of mDA markers TH, NURR1, and PITX3 at day 42 of differentiation from MShef7 and RC17 hESC lines (n = 3 experimental replicates).


More Than 25 Tips For Gaining Well-cultured Cells

Cell culture is not an easy thing which needs more care. Paying more attention to following tips, you will do it better.

Tip 1: How to thaw cooling tubes?
Cooling tubes should be put into the 37 ℃ water tank immediately for rapid thaw after taking out, shaking to melt within 1 minute. They key point is t keep the water level lower than cooling tubes’ covers in order to avoid pollution. One more thing, after taking out cooling tubes from liquid nitrogen bucket, extra concern should be needed to prevent the burst for your safety.

Tip 2: Should DMSO be removed immediately while thawing?
Except for a small number of cells that have been particularly specified to be sensitive to DMSO, most cell lines (including suspension cells), after thawing, should be placed directly into the cell culture flask with 10

15 ml fresh medium, and we should replace the medium the next day in order to remove DMSO, and then most of the thawed cells’ growing or sticking problems can be avoided.

Tips 3: Can I use mediums with different culture conditions from original ones ?
Absolutely not! Each cell line has its specific and adapted cell culture medium, mediums with different culture conditions, to which most cells can not adapt immediately, will result in cells death.

Tip 4: Can I use serum with different types from original one?
Of course not! Serum is a very important source of nutrition in cell culture, its type and quality will have a great impact on cell growth. Different species have different substances or molecular amount in serum. Inappropriate serum will cause cell survival problem.

Tip 5: What is FBS, FCS, CS, HS?
FBS (fetal bovine serum) and FCS (fetal calf serum) share the same meaning—fetal bovine serum. FCS, an incorrect expression, should not be used. CS (calf serum) stands for calf serum and HS (horse serum) for horse serum.

Tip 6: Should I use 5% CO2 or 10% CO2 when culturing cells? Or have no effect at all?
Generally speaking, HCO3- / CO32- / H + is used as the pH buffer system in most of the culture medium, and the content of NaHCO3 in the culture medium will determine the CO2 concentration while culturing. When NaHCO3 content is 3.7 g/liter in the medium, 10% CO2 is needed when its content is 1.5 g/liter, 5% CO2 is applied.

Tip 7: When should I change the medium? Is it necessary to add antibiotics in the medium?
This depends on cell growth density, or changing medium on time according to the replacement time on the basic data of the cell line. Under normal culture state, no antibiotics should be added to the culture medium, except in a special screening system.

Tip 8: What is the trypsin-EDTA concentration when the adherent cells are subcultured? How to handle it?
Generally-used trypsin-EDTA concentration is 0.05% trypsin-0.53 mM EDTA.4 Na. After opening the bottle for the first time, trypsin-EDTA should be stored respectively in sterile test tubes in a small amount at -20 ℃, to do so, we can avoid trypsin activity decreased caused by repeated freezing and thawing and reduce the risk of pollution.

Tip 9: What are the subculture processes for suspended cells?
Generally, what we only to do is to adding fresh culture medium to the original culture bottle continuously to dilute the cell concentration. If the culture medium is too much, we can slightly raise the mouth of the culture bottle until it can not be accommodated. Transferring a part of the cell-containing culture medium to another new culture flask when you are separating the culture medium, and adding fresh medium to dilute to the appropriate concentration, repeating the previous steps.

Tip 10: How much rotating speed should be to centrifuge general animal cells?
The centrifugal rate is generally 1,000 rpm and 5 to 10 minutes to recover animal cells. Excessive speed will cause cell death.

Tip 11: What is the inoculated density of cells?
Inculated density can be in accordance with the density of cell lines’ basis data or the proportion of dilution. The small number of cells or diluting too much is also one of the important negative causes of cell growth.

Tip 12: What is the composition of cell freezing medium?
The most commonly-used fresh frozen medium for cryopreserving animal cells contains 5%

10% DMSO (dimethyl sulfoxide) and 90%

95% medium for the original cell growth mixed evenly.

Tip 13: What is the level of DMSO and the way of sterile filtration?
DMSO grade for frozen storage must be tissue culture grade, and it is sterile itself. After opening the bottle for the first time, you should store DMSO respectively in sterile test tubes in a small amount at 4 ℃, and then harmful substances released by DMSO cleavage through repeated freezing and thawing can be avoided, and also the risk of pollution will be reduced. And you can use DMSO-resistant Nylon filter to filter DMSO.

Tip 14: How to cryopreserve cells? And what is the cell concentration in cooling tubes during cryopreservation?
Cryopreservation method A: placing cooling tubes at 4 ℃ for 30

60 minutes → (-20 ℃ for 30 minutes) →-80 ℃ for 16

18 hours (or overnight) →liquid nitrogen tank vapor phase for long-term storage

Cryopreservation method B: placing cooling tubes in the automatic cooling machine, whose program has been set to drop 1

3 ℃ per minute, until to -80 ℃ or below. And then you should place it into liquid nitrogen tank vapor phase for long-term storage. Storing at -20 ℃ can not be more than 1 hour in order to prevent too large ice crystals and a large number of dead cells. This step can also be ignored, and placing tubes into -80 ℃ refrigerator and lowering survival rate slightly.

The number of cells in the cooling tube is generally 1𴡂 cells/ml vial, and that of fusion tumor cells is preferably 5𴡂 cells/ml vial.

Tip 15: How to avoid cell contamination? How to deal with microbial contamination?
Cell contamination can be classified into bacteria, yeasts, molds, viruses and mold bacteria. The main causes of contamination are improper operation of aseptic technique, poor environment of operating room, contaminated serum, contaminated cells and so on. The best ways to reduce cell contamination are mainly depending on strict aseptic technique, clean environment, well-qualified cell sources and medium preparation.

When microbial contamination occurs, we should add appropriate antibiotics, sterilize and discard cells directly.

Tip 16: Can I observe the abnormal state of mycoplasma contaminated cells with naked eyes and its effect on the cell culture?
We can not observe the abnormal state of mycoplasma contaminated cells with naked eyes. Except very experienced experts, general researchers can not distinguish most mycoplasma contaminated cell lines according to their appearance.

Mycoplasma contamination can affect almost all cell growth parameters, metabolize and any other study data. Therefore, before the experiment performed, we must confirm that the cells are mycoplasma-free, and then the experimental result data can be meaningful.

Mycoplasma contaminated cells lines should be directly sterilized and discarded to avoid contaminating other cell lines.

Tip 17: Why the medium color will be dark red and the pH will be more and more alkaline when stored in the refrigerator at 4 ℃?
When storing in the refrigerator at 4℃, CO2 in the culture medium will gradually overflow, and this will cause the medium get more and more alkaline, the color of the acid-base indicator (usually phenol red) in the culture medium will also be darker with the alkaline and then become dark red. Alkaline culture medium will result in cell growth stagnation or death. Filtering CO2 aseptically to adjust the pH value should be a proper method when culture medium gets alkaline.

Tip 18: Are the dish and flask same for all kinds of cell culture?
Dishes or flasks from different brands coat different polymer and have different manufacturing processes. Although there is no great impact on most cells, only a few cells may have growth differences due to the use of dishes or flasks from different brands.

Tip 19: Possible causes for cell death or poor survival rate? After the cell freezing tube thawed, why are there a small number of cells?
Common causes for cells’ poor survival rate: improper use of medium or poor quality of medium improper use of serum or poor-quality serum thawing process error washing and centrifuging frozen cells after thawing misreading suspension cells as dead cells improper culturing temperature long-time storage at -80 ℃.

The small number of cells in the frozen cell culture is mostly due to error operation of the centrifugal, this results in cells’ physical damage and cell loss. Do not immediately centrifuge the cells after thawing, and should replace medium overnight after cell growth.

Tip 20: Reasons for rupture frozen tube bottle, cracked cap or fall-off cap?
Rupture frozen tube bottle and cracked cap may be due to the improper force when operator held the frozen tube. The hemostatic clamp is recommended to use and held carefully. As for fall-off caps, it is because of the physical phenomenon—heat makes it expand and cold makes it contract, and cooling tubes may be contaminated. So whether it is put into or taken out of the liquid nitrogen bucket, cooling tubes should be immediately tightened.

Tip 21: How to choose a special cell line culture medium?
There is no fixed culture condition for a certain cell type. Cells cultured in MEM are likely to grow well in DMEM or M199 as well. In a word, it will be a good start to choose MEM for adherent cell culture, RPMI-1640 for suspension cell culture. The best serum-free medium for various purposes prefers AIM V (12005) culture medium (SFM).

Tip 22: Is L-glutamine important in cell culture? Is it unstable in solution?
L-glutamine plays a very crucial role in cell culture. After removal of the amino group, L-glutamine can be used as a source of energy for cultured cells, involved in protein synthesis and nucleic acid metabolism. L-glutamine will be degraded in solution in a period of time, but the exact degradation rate has not been finalized. The degradation of L-glutamine leads to the formation of ammonia, and the ammonia is toxic to some cells.

Tip 23: What is GlutaMAX-I? How to use GlutaMAX-I in cell culture? How about its stability?
The GlutaMAX-I dipeptide is a derivative of L-glutamine, and its unstable alpha-amino group is protected by L-alanine. A certain peptidase gradually cleaves the dipeptide to release GlutaMAX-I for use. GlutaMAX-I dipeptide is very stable, it degrades a little even sterilized at 121 pounds for 20 minutes. Under the same condition, L-glutamine is almost completely degraded.

Tip 24: What medium can work well without phenol red? What is the role of sodium pyruvate in the culture medium?
We always use Phenol red as a indicator of pH in the medium: it will be red when neutral, yellow when acidic and purple when alkaline. Studies have shown that phenol red can mimic the role of steroid hormones (especially estrogen). In order to avoid the steroid reaction, we should culture cells especially mammalian cells in no phenol red medium. And since the phenol red interfers detection, some researchers use mediums without phenol red to do flow cytometry.

Sodium pyruvate can be used as an alternative carbon source in cell culture. Although cells tend to use glucose as a carbon source, cells can also metabolize sodium pyruvate if there is no glucose.

Tip 25: Why does not Hank’s balanced salt solution (HBS) used in the air need for CO2 incubators? What are the essential functional differences between HBS and Earle’s balanced salt solution (EBS)?
The main difference between HBS and EBS is the level of sodium bicarbonate, and it is much higher in Eagles (2.2 g/L) than in Hanks (0.35 g/L). Sodium bicarbonate requires a high level of CO2 to maintain the pH of the solution. Eagles liquid will become alkali in the air level of CO2, hanks solution in the CO2 incubator will become acidic. We can choose eagles solution for storing tissue in a CO2 incubator, and hanks solution for cleaning tissue stored in the cell culture medium.

Tip 26: Does divalent ions inhibit trypsin activity? What is the purpose of adding EDTA when using trypsin?
Divalent ions can indeed inhibit trypsin activity. EDTA is used to chelate free magnesium ions and calcium ions in order to maintain inhibition of trypsin activity. So it is recommended that cells be washed with EDTA to remove all divalent ions from the medium before dealing the cells with trypsin.

Tip 27: Other precautions
1.We should apply at most 50% concentration of antibiotics used in serum medium to serum-free medium. Serum proteins will combine and inactivate some antibiotics. Under the serum-free culture conditions, if antibiotics are not inactivated and they may be toxic to cells.
2.Once you add serum and antibiotics to fresh medium, you should used it within two to three weeks. Because some basic components of the antibiotics and serum will begin to degrade after thawing.
3.Most of the additives and reagents can be repeatedly frozen up to 3 times at most, many times of operation will cause a certain level of degradation and precipitation in protein solution, and this will affect its performance.
4.Within a week after dissolution, we should use the liquid trypsin solution stored in a refrigerator at 4 ℃. Trypsin may begin to degrade at 4 ℃ and become unstable at room temperature for more than 30 minutes.
5.In order to keep the water tray in the CO2 incubator clean, you must replace (at least once every two weeks) with it sterile distilled water or sterile deionized water.

All above are some tips for cell culture. If you got another tips, you can discuss with us.


Affiliations

Technische Universitaet Muenchen (TUM), Munich, Germany

Lehrstuhl für Vergleichende Tropenmedizin und Parasitologie, Ludwig-Maximilians-Universitaet Muenchen, Leopoldstr. 5, 80802, Munich, Germany

Erich Zweygarth & Lygia MF Passos

The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, UK

Departamento de Medicina Veterinária Preventiva, Escola de Veterinária- UFMG, Belo Horizonte, Minas Gerais, Brazil

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Corresponding author


ACKNOWLEDGMENTS

We thank Ms Junko Hirao and Ms Eriko Komaki for their technical assistance, and Dr Yukihiro Nagatani at Hikone Municipal Hospital, Shiga, Japan, for preparing archival tissue samples at the initial diagnosis of the index patient. The experiments with IVIS were conducted through the Joint Usage/Research Center Program of the Radiation Biology Center, Kyoto University. This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and by a research grant from the Japanese Foundation for Prostate Research.


Invitromatics, invitrome, and invitroomics: introduction of three new terms for in vitro biology and illustration of their use with the cell lines from rainbow trout

The literature on cell lines that have been developed from rainbow trout (RT) (Oncorhynchus mykiss) is reviewed to illustrate three new terms: invitromatics, invitrome, and invitroomics. Invitromatics is defined as the history, development, characterization, engineering, storage, and sharing of cell lines. RT invitromatics differs from invitromatics for humans and other mammals in several ways. Nearly all the RT cell lines have developed through spontaneous immortalization. No RT cell line undergoes senescence and can be described as being finite, whereas many human cell lines undergo senescence and are finite. RT cell lines are routinely grown at 18–22°C in free gas exchange with air in basal media developed for mammalian cells together with a supplement of fetal bovine serum. An invitrome is defined as the grouping of cell lines around a theme or category. The broad theme in this article is all the cell lines that have ever been created from O. mykiss, or in other words, the RT invitrome. The RT invitrome consists of approximately 55 cell lines. These cell lines can also be categorized on the basis of their storage and availability. A curated invitrome constitutes all the cell lines in a repository and for RT consists of 11 cell lines. These consist of epithelial cell lines, such as RTgill-W1, and fibroblast cell lines, such as RTG-2. RTG-2 can be purchased from a scientific company and constitutes the commercial RT invitrome. Cell lines that are exchanged between researchers are termed the informally shared invitrome and for RT consists of over 35 cell lines. Among these is the monocyte/macrophage cell line, RTS11. Cell lines whose existence is in doubt are termed the zombie invitrome, and for RT, approximately 12 cell lines are zombies. Invitroomics is the application of cell lines to a scientific problem or discipline. This is illustrated with the use of the RT invitrome in virology. Of the RT invitrome, RTG-2 was the most commonly used cell line to isolate viruses. Fifteen families of viruses were studied with RT invitrome. RT cell lines were best able to support replication of viruses from the Herpesviridae, Iridoviridae, Birnaviridae, Togaviridae, and Rhabdoviridae families.

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