While addition of L-ascorbic acid (Asc) only marginally enhanced cell growth, trisodium L-ascorbic acid 2-phosphate (0.1-1.5 mM; media changes every 2 to 3 days for 2 to 3 weeks) greatly stimulated cell growth. stimulation of growth. Cell growth is greatly enhanced by the combination of Asc-2P and bFGF; however, additional effects are not produced by the addition of EGF or insulin [1]. Higher doses of AsA2-P lead to enhanced runx2 expression and ALP activity. L-ascorbic acid 2-phosphate (50 µM-250 µM) trisodium is required for the efficient osteogenic differentiation of human adipose stem cells (hASC). The combinations of 150 µM asA2-P and 10 nM dexamethasone (Dex) and 250 µM asA2-P and 5 nM Dex produced the maximum proliferation, ALP activity, and runx2 expression [3].

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In human corneal endothelial cells (HCECs), Asc-2P at 0.06–0.3 mM (optimal range, up to 1.5 mM) significantly stimulated cell growth, while ascorbic acid showed only weak stimulation. Combination of Asc-2P (0.3 mM) with bFGF (2 ng/mL) on atelocollagen promoted HCEC proliferation in both primary cultures and subcultures. Asc-2P extended the replicative lifespan of HCECs: cells cultured without Asc-2P underwent senescence at population doubling level (PDL) 27.3, whereas cells with Asc-2P did not show senescence over PDL 40. Asc-2P reduced oxidative DNA damage: 8-hydroxy-2-deoxyguanosine (8-OHdG) levels in mitochondrial DNA were significantly lower when HCECs were subcultured with Asc-2P compared to without. [1]
- In human skin fibroblasts, Asc-2P (0.2 mM) alone raised production of type I collagen, increased the steady-state levels of proα1(I) and proα2(I) collagen mRNAs (to 131±4% and 127±6% of control respectively), and stimulated transcriptional activity of these genes. When co‑present with human EGF (10 ng/mL), Asc-2P attenuated EGF’s inhibitory effects on collagen production, mRNA levels, and gene transcription. [2]
- In human adipose stem cells (hASCs), higher concentrations of AsA2-P (150–250 μM) combined with lower dexamethasone (10–5 nM) enhanced osteogenic differentiation. Specifically, in human serum‑based medium, AsA2-P at 250 μM with 5 nM dexamethasone (OM3) significantly increased alkaline phosphatase (ALP) activity (P=0.036 vs. maintenance medium) and mineralization. AsA2-P at 150 μM with 10 nM dexamethasone (OM2) also promoted mineralization. In FBS‑based medium, osteogenic differentiation was weaker, but upregulation of osteogenic marker genes (runx2A, collagen type I, ALP) was observed with AsA2-P containing media. In defined xeno‑free RegES medium, plain RegES already contained 170 μM AsA2-P, supporting early osteogenic differentiation, though additional AsA2-P and dexamethasone were needed for mineralization. [3]

Human corneal endothelial cells (HCECs): HCECs from donors aged 12–74 years were isolated, seeded on atelocollagen‑coated dishes, and cultured in DMEM with 15% FCS and bFGF (2 ng/mL) with or without Asc-2P (0.3 mM). Primary cultures with Asc-2P reached confluence within 2–3 weeks, while most without Asc-2P showed poor growth. Cell proliferation was assessed by cell counting after trypsinization. Population doubling level (PDL) was calculated as PDL = log(X1/X0)/log2. For 8-OHdG detection, HCECs subcultured five times with or without Asc-2P were immunostained with anti-8-OHdG antibody. For quantification, mitochondrial DNA was isolated, digested with nuclease P1 and alkaline phosphatase, and 8-OHdG levels measured by competitive ELISA (range 0.125–10 ng/mL). Results showed significantly lower 8-OHdG in Asc-2P‑treated cells. Immunostaining for ZO-1, N-cadherin, connexin 43, and Na+/K+-ATPase showed proper localization in cells cultured with Asc-2P. RT-PCR detected VDAC2, VDAC3, CLCN2, CLCN3, SLC4A4, and AQP1 mRNAs. [1]
- Human skin fibroblasts: Fibroblasts from normal adults were cultured in DMEM with 10% FBS. Cells were treated with human EGF (10 ng/mL) and/or Asc-2P (0.2 mM) for up to 14 days. For collagen production, cells were labeled with [3H]proline for the last 2–24 h, and collagenase‑sensitive and ‑resistant proteins were quantified. Relative collagen production was calculated assuming collagen has 5.4 times higher proline content. Collagen types were analyzed by SDS‑PAGE (5% gel) with or without DTT, followed by fluorography and densitometry. For mRNA analysis, total RNA was extracted and dot‑blot or Northern blot hybridized with 32P‑labeled cDNAs for proα1(I), proα2(I), and β‑actin. Asc-2P increased proα1(I) and proα2(I) mRNA levels to 131% and 127% of control, respectively. Nuclear run‑off transcription assays were performed: nuclei isolated from treated cells were incubated with [α-32P]NTPs for 10 min (linear incorporation), labeled RNA hybridized to cDNA spots, and transcriptional activity calculated as (specific counts - background)/(β‑actin counts - background). Asc-2P increased transcriptional activity of type I collagen genes. [2]
- Human adipose stem cells (hASCs): hASCs isolated from subcutaneous adipose tissue were cultured in maintenance medium (DMEM/F-12 with 10% FBS or 10% human serum). For osteogenic differentiation, cells were seeded at 7×10³ cells/cm², and after 24 h, medium was replaced with osteogenic media containing different concentrations: OM1 (50 μM AsA2-P + 100 nM dexamethasone + 10 mM β‑glycerophosphate), OM2 (150 μM AsA2-P + 10 nM dexamethasone + 10 mM β‑GP), OM3 (250 μM AsA2-P + 5 nM dexamethasone + 10 mM β‑GP). At days 7 and 14, ALP activity was measured colorimetrically and normalized to DNA content (CyQUANT assay). Mineralization was detected by Alizarin Red S staining and quantified by cetylpyridinium chloride extraction (absorbance at 540 nm). qRT-PCR analyzed expression of runx2A, DLX5, collagen type I, osteocalcin, and ALP, normalized to RPLP0. In human serum‑based medium, OM3 significantly increased ALP activity (P=0.036 vs. MM; P=0.006 vs. OM1) and mineralization (OM2 and OM3 vs. MM and OM1). In FBS‑based medium, OM3 upregulated runx2A mRNA at day 14 (P=0.048 vs. MM). In RegES medium (containing 170 μM AsA2-P base), all OMs increased mineralization vs. MM. [3]

In HCECs, Asc-2P showed no or almost no toxicity at concentrations up to 1.5 mM. In contrast, high concentration (1 mM) of ascorbic acid had strong cytotoxic effects, inducing apoptosis. Asc-2P is resistant to auto‑oxidation in culture medium and does not produce ascorbyl radicals, whereas ascorbic acid rapidly oxidizes under ambient oxygen (21% O2) and generates reactive oxygen species (mainly H2O2). [1]
- In human skin fibroblasts, Asc-2P at 0.2 mM was well tolerated and stimulated cell growth and protein production without apparent cytotoxicity. [2]
- In hASCs, Asc-2P at concentrations up to 250 μM (with 5 nM dexamethasone) supported cell proliferation and differentiation without reported toxicity. The defined RegES medium containing 170 μM Asc-2P also supported robust cell growth. [3]

[1]. Increased proliferation and replicative lifespan of isolated human corneal endothelial cells with L-ascorbic acid 2-phosphate.Invest Ophthalmol Vis Sci. 2011 Nov 7;52(12):8711-7.

[2]. Epidermal growth factor inhibits transcription of type I collagen genes and production of type I collagen in cultured human skin fibroblasts in the presence and absence of L-ascorbic acid 2-phosphate, a long-acting vitamin C derivative.J Biol Chem. 1991 May 25;266(15):9997-10003.

[3]. Effects of different serum conditions on osteogenic differentiation of human adipose stem cells in vitro.Stem Cell Res Ther. 2013 Feb 15;4(1):17.

Asc-2P is a long‑acting, oxidation‑resistant derivative of L‑ascorbic acid (vitamin C). It is dephosphorylated to free ascorbic acid by alkaline phosphatase on the cell membrane, thereby delivering a high intracellular dose of vitamin C without generating cytotoxic ascorbyl radicals. [1][2]
- In human corneal endothelial cells, Asc-2P extends replicative lifespan partly by protecting against oxidative DNA damage, as evidenced by reduced 8-OHdG levels in mitochondrial DNA. The normal concentration of ascorbic acid in the human anterior chamber is approximately 500 μM, suggesting physiological relevance. [1]
- In human skin fibroblasts, Asc-2P and EGF competitively regulate transcription of type I collagen genes, suggesting a role for Asc-2P in maintaining collagen homeostasis. [2]
- In human adipose stem cells, the commonly used osteogenic medium (OM1, 50 μM AsA2-P/100 nM dexamethasone) was less effective than formulations with higher AsA2-P (150–250 μM) and lower dexamethasone (10–5 nM). Serum conditions (FBS vs. human serum vs. xeno‑free) significantly affect osteogenic differentiation. The xeno‑free RegES medium contains 170 μM AsA2-P, enabling early osteogenic induction. [3]

C6H6NA3O9P

322.05

321.944

66170-10-3

L-Ascorbic acid 2-phosphate magnesium;113170-55-1;L-Ascorbic acid 2-phosphate;23313-12-4

10990876

White to off-white solid powder

84 °C

2

9

3

19

357

2

C([C@@H]([C@@H]1C(=C(C(=O)O1)OP(=O)([O-])[O-])[O-])O)O.[Na+].[Na+].[Na+]

YRWWOAFMPXPHEJ-OFBPEYICSA-K

InChI=1S/C6H9O9P.3Na/c7-1-2(8)4-3(9)5(6(10)14-4)15-16(11,12)13;;;/h2,4,7-9H,1H2,(H2,11,12,13);;;/q;3*+1/p-3/t2-,4+;;;/m0.../s1

sodium (R)-5-((S)-1,2-dihydroxyethyl)-2-oxido-4-oxo-4,5-dihydrofuran-3-yl phosphate

2-Phospho-L-ascorbic acid trisodium salt VOK-70103 VOK70103VOK 70103523114

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~150 mg/mL (~465.77 mM)
DMSO : ~6 mg/mL (~18.63 mM)

Solubility in Formulation 1: 120 mg/mL (372.61 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)