CN115125202A - 一种自体血细胞因子和外泌体血清及其制备方法 - Google Patents
一种自体血细胞因子和外泌体血清及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种自体血细胞因子和外泌体血清及其制备方法。所述制备方法包括:对外周血进行第一离心处理,分离得到红细胞与血浆层,所述血浆层包含血小板和白细胞;对所述血浆层进行第二离心处理,使血小板和白细胞沉降,并形成细胞团;以三联激活物对所述细胞团进行三联激活,之后温控孵育,释放出细胞因子和外泌体,并纯化,得到自体血细胞因子和外泌体血清。本发明提供的自体血细胞因子和外泌体血清的制备方法通过两步分离法提升了传统PRP/PRF等血小板获得率与浓缩比并且降低生产成本,结合三联激活与温控孵育,提升了血小板释放细胞因子的释放度与最终浓度,增加了免疫因子与外泌体的释放,同时可减轻皮下注射疼痛感。
Description
技术领域
本发明涉及一种制备细胞因子和外泌体血清的方法,具体涉及一种自体血细胞因子和外泌体血清及其制备方法,属于生物技术领域。
背景技术
血液细胞如血小板、白细胞在创伤的愈合和细胞的增殖与分化及组织的形成有着极其重要的作用。1882年意大利医师J.B.比佐泽罗发现血小板;1977年Harkedeng首次分离制备出PRP,将其用于心外科手术后患者,取得良好的疗效。1984年Okuda等发现血浆中提取的富血小板血浆含多种生长因子。1993年Hood等在PRP中加入凝血酶和钙离子,提出了PG概念,将其所形成的凝胶状物质用于组织修复。1997年Whitman率先将自体PRP用于口腔颌面外科手术修补骨组织。2003年伦敦Kubota和Otto医生对PRP制作流程和应用基础技术的基础上开发了全新的PRP自体细胞提取技术,规避了PRP原有的缺陷。目前,这项技术已经获得美国FDA、欧洲CE认证及其他地区的广泛医学临床验证,国外PRP技术已经得到广泛的应用。国内深圳与北京已将该技术纳入新增医疗服务体系中,逐渐在多个科室如骨科、烧伤整形科、牙科、外科、疼痛科等应用。
但目前PRP技术存在仍三个问题需要进一步改善。其一是目前PRP制备技术血小板回收率较低,往往低于80%,且白细胞丢失,导致免疫因子含量低,从而降低了PRP抗炎与免疫驱化效果。其二是注射后有较明显的刺痛感,多因为PRP为血浆复合物,含有大量纤维蛋白原,血小板处于未激活状态,注射时接触体内胶原蛋白从而引起血小板聚集激活,并释放如前列腺素等物质引起剧烈疼痛,从而影响患者的体验感,产生恐惧感乃至抵触继续进行该技术后续的使用。其三,注射PRP在72小时后,细胞因子外泌体释放曲线才到顶点,但注射后24小时内血小板已经被体内巨噬细胞吞噬从而导致细胞因子、外泌体的释放不完全,不能充分发挥PRP的潜力。
发明内容
本发明的主要目的在于提供一种自体血细胞因子和外泌体血清及其制备方法,以克服现有技术中的不足。
为实现前述发明目的,本发明采用的技术方案包括:
本发明实施例提供了一种自体血细胞因子和外泌体血清的制备方法,其包括:
提供外周血,对所述外周血进行第一离心处理,分离得到红细胞与血浆层,所述血浆层包含血小板和白细胞;
对所述血浆层进行第二离心处理,使血小板和白细胞沉降,并形成细胞团;
以三联激活物对所述细胞团进行三联激活,之后温控孵育,释放出细胞因子和外泌体,并纯化,得到自体血细胞因子和外泌体血清。
在一些优选实施例中,所述三联激活物包括钙剂、氨甲环酸和ATP。
本发明实施例提供了前述的制备方法制得的高浓度的自体血细胞因子和外泌体血清。
与现有技术相比,本发明的优点包括:
本发明提供的自体血细胞因子和外泌体血清的制备方法通过两步分离法提升了传统PRP/PRF等血小板获得率与浓缩比并且降低生产成本,结合三联激活与温控孵育,提升了血小板释放FGF、EGF、PDGF、TGF-β等细胞因子的释放度与最终浓度,增加了免疫因子与外泌体的释放,同时可减轻皮下注射疼痛感。
附图说明
为了更清楚地说明本发明的技术方案,下面将对实施例或现有技术描述中所需要使用的附图进行简单的介绍,显而易见地,下面描述的附图仅仅作为本文发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他附图。
图1为本发明一典型实施例中制备得到的含多种高浓度的细胞因子与外泌体的血清产物中各因子浓度检测对比示意图。
图2是本发明一典型实施例中不同温度下进行实验的对比示意图。
具体实施方式
基于以上分析,本案发明人经长期研究和大量实践,得以提出本发明的技术方案,其建立了一种自体血细胞因子和外泌体的血清制备方法,主要是采用两步离心分离法、三联激活、温控孵育等方法将上述问题改善。如下将对该技术方案、其实施过程及原理等作进一步的解释说明。
本发明实施例的一个方面提供的一种自体血细胞因子和外泌体血清的制备方法,主要包括:采用两步离心分离法分离外周血白细胞、血小板并使用三联激活与温控孵育血小板、白细胞以产生丰富的自体血细胞因子和外泌体,再纯化出高浓度的自体血细胞因子和外泌体血清。
在一些实施例中,所述制备方法包括:
提供外周血,对所述外周血进行第一离心处理,分离得到红细胞与血浆层,所述血浆层包含血小板和白细胞;
对所述血浆层进行第二离心处理,使血小板和白细胞沉降,并形成细胞团;
以三联激活物对所述细胞团进行三联激活,之后温控孵育,促进血细胞与白细胞的细胞因子和外泌体的释放,并纯化,得到自体血细胞因子和外泌体血清。
在一些优选实施例中,所述制备方法包括:采用冷冻离心机进行所述第一离心处理,离心力为400~450g,离心处理的时间为5min,所述冷冻离心机的温度为4~8℃。
在一些优选实施例中,所述制备方法包括:采用冷冻离心机进行所述第二离心处理,离心力为1400~1500g,离心处理的时间为7~10min,所述冷冻离心机的温度为4~8℃。
进一步地,在所述第二离心处理结束后,血小板和白细胞的回收率在95%以上。
本发明的两步离心分离法主要是:第一步,低离心力使血液分层,分为红细胞层和血浆层,吸取血浆层。第二步高离心力离心血浆,使血小板和白细胞沉降。
本发明采用两步分离法可提升传统PRP/PRF等血小板获得率与浓缩比并且降低生产成本。并且,使用两步离心分离法,可使血小板和白细胞回收率提高至95%以上,且成本较低。
在一些优选实施例中,所述三联激活物包括钙剂、氨甲环酸和ATP。其中,钙剂为血小板致聚剂,单独使用可使血细胞板凝集。本发明使用钙剂、氨甲环酸、ATP三联激活物,相较单独使用钙剂,可促使血小板聚集,同时维持血小板、白细胞较长时间存活,使两者在温控孵育的条件下,释放出大量细胞因子,外泌体。
进一步地,所述钙剂、氨甲环酸与ATP的摩尔比为6~12∶10~20∶0.1~0.2。
在一些优选实施例中,所述温控孵育的温度为37.5~38℃,孵育时间为48~72h。本发明采用的孵育温度高于正常体温是因为血管破损出血后血小板形成凝血块止血后,召集白细胞到伤口处形成局部炎症。此时局部温度较高,可达到37.5~38℃,更有利于白细胞、血小板合成与释放大量细胞因子与外泌体。
本发明同时采用三联激活与温控孵育,可提升血小板释放FGF、EGF、PDGF、TGF-β等细胞因子的释放度与最终浓度,增加免疫因子与外泌体的释放,同时减轻皮下注射疼痛感。
在一些优选实施例中,所述制备方法还包括:将所述自体血细胞因子和外泌体血清于4~8℃冷藏48~72h,之后再进行第三离心处理,获得高浓度的自体血细胞因子和外泌体血清。此阶段为冷藏释放期,期间细胞凝集团仍在缓慢释放细胞因子,可增加细胞因子的最终产量。另外4~8℃可保持各种细胞因子与外泌体活性。
在一些优选实施例中,所述制备方法还包括:采用冷冻离心机进行所述第三离心处理,离心力为1400~1500g,离心处理的时间为10~15min。
本发明实施例的另一个方面提供了由前述制备方法制得的高浓度的自体血细胞因子和外泌体血清。
其中,所述细胞因子可以是FGF、EGF、PDGF、TGF-β等,但不限于此。
藉由上述技术方案,本发明提供的自体血细胞因子和外泌体血清的制备方法通过两步分离法提升了传统PRP/PRF等血小板获得率与浓缩比并且降低生产成本,结合三联激活与温控孵育,提升了血小板释放细胞因子的释放度与最终浓度,增加了免疫因子与外泌体的释放,同时可减轻皮下注射疼痛感。
为使本发明的目的、技术方案和优点更加清楚,下面结合若干优选实施例并结合附图对本发明的技术方案进行进一步具体描述,但本发明并不仅仅局限于下述实施例,该领域技术人员在本发明核心指导思想下做出的非本质改进和调整,仍然属于本发明的保护范围。若非特别说明,则下列实施例中使用的各种试剂均是本领域技术人员熟知的,并可以通过市场购买等途径获取。而下列实施例中未注明具体条件的实验方法,通常按照常规条件或按照制造厂商所建议的条件。
实施例1
本实施例采用的自体血细胞因子和外泌体血清的制备方法具体如下:
1、使用无菌EDTA抗凝管采集外周血60毫升。
2、冷冻离心机(温度设置为4~8℃)分离抗凝血,采用400~450g离心力,离心5min。一方面低温可以减少血小板的机械力激活,另一方面400~450g低离心力情况下,可使红细胞与血浆分层,而血浆层保留绝大部分白细胞与血小板。
3、用无菌吸管将血浆小心吸取,转移至无菌离心管中,尽量不吸取红细胞,得血浆体积在25~35mL不等,此步白细胞与血小板的回收率可达95%以上。
4、冷冻离心机(温度设置为4~8℃)中离心分离上述收集的血浆,1400~1500g离心力,7~10min;此离心力与离心时间可使白细胞与血小板几乎完全沉降,形成细胞团,此时白细胞与血小板回收率仍可达到95%以上。
5、去除大部分上层血浆,剩余5~10mL底层血浆与离心管底部的白细胞,血小板的细胞团,浓缩比可达6~12倍。
6、向细胞团与剩余血浆加入三联激活物并震荡均匀,三联激活物为钙剂、氨甲环酸、ATP,其中,所述钙剂、氨甲环酸与ATP的摩尔比可以是6~12∶10~20∶0.1~0.2。钙剂、氨甲环酸可使血小板聚集,激活,ATP可延长白细胞与血小板的存活时间,存活时间可达到96小时。将细胞悬液放入培养箱中,温度设置到37.5~38℃,持续孵育48~72小时,纤维蛋白原被激活发生收缩,细胞悬液将凝集成团。孵育温度高于正常体温是因为血管破损出血后血小板形成凝血块止血后,召集白细胞到伤口处形成局部炎症。此时局部温度较高,可达到37.5~38℃,更有利于白细胞、血小板合成与释放大量细胞因子与外泌体;另外,本案发明人还对低于该温度的情形进行了对照实验(温度设置为36.8℃),结果如图2所示。血在单纯同浓度的钙剂、氨甲环酸、ATP与同浓度的钙剂加氨甲环酸、ATP两条件下,小板与白细胞存活时间与存活率对比如下表所示:
7、孵育48~72小时后,将其放入4~8℃冰箱冷藏48~72小时天,此阶段为冷藏释放期,期间细胞凝集团仍在缓慢释放细胞因子,可增加细胞因子的最终产量。另外4~8℃可保持各种细胞因子与外泌体活性。而前列腺素半衰期仅20min,因此经过孵育期与冷藏释放期,已经失活。冷藏释放期后,置入冷冻离心机分离,1400~1500g离心力,离心10min,可得到含多种高浓度的细胞因子与外泌体的、无纤维蛋白原的、无前列腺素(PG)的血清产物。其中各因子浓度检测对比如图1所示。
综上所述,本发明通过两步分离法提升了传统PRP/PRF等血小板获得率与浓缩比并且降低生产成本,结合三联激活与温控孵育,提升了血小板释放细胞因子的释放度与最终浓度,增加了免疫因子与外泌体的释放,同时可减轻皮下注射疼痛感。
应当理解,以上所述的仅是本发明的一些实施方式,应当指出,对于本领域的普通技术人员来说,在不脱离本发明的创造构思的前提下,还可以做出其它变形和改进,这些都属于本发明的保护范围。
Claims (10)
1.一种自体血细胞因子和外泌体血清的制备方法,其特征在于包括:
提供外周血,对所述外周血进行第一离心处理,分离得到红细胞与血浆层,所述血浆层包含血小板和白细胞;
对所述血浆层进行第二离心处理,使血小板和白细胞沉降,并形成细胞团;
以三联激活物对所述细胞团进行三联激活,之后温控孵育,释放出细胞因子和外泌体,并纯化,得到自体血细胞因子和外泌体血清。
2.根据权利要求1所述的制备方法,其特征在于,包括:采用冷冻离心机进行所述第一离心处理,离心力为400~450g,离心处理的时间为5min,所述冷冻离心机的温度为4~8℃。
3.根据权利要求1所述的制备方法,其特征在于,包括:采用冷冻离心机进行所述第二离心处理,离心力为1400~1500g,离心处理的时间为7~10min,所述冷冻离心机的温度为4~8℃。
4.根据权利要求3所述的制备方法,其特征在于:在所述第二离心处理结束后,血小板和白细胞的回收率在95%以上。
5.根据权利要求1所述的制备方法,其特征在于:所述三联激活物包括钙剂、氨甲环酸和ATP,并且,所述钙剂、氨甲环酸与ATP的摩尔比为6~12∶10~20∶0.1~0.2。
6.根据权利要求1所述的制备方法,其特征在于:所述温控孵育的温度为37.5~38℃,孵育时间为48~72h。
7.根据权利要求1所述的制备方法,其特征在于,还包括:将所述自体血细胞因子和外泌体血清于4~8℃冷藏48~72h,之后再进行第三离心处理,获得高浓度的自体血细胞因子和外泌体血清。
8.根据权利要求7所述的制备方法,其特征在于,包括:采用冷冻离心机进行所述第三离心处理,离心力为1400~1500g,离心处理的时间为10~15min。
9.由权利要求1-8中任一项制备方法制得的自体血细胞因子和外泌体血清。
10.根据权利要求9所述的自体血细胞因子和外泌体血清,其特征在于:所述细胞因子包括FGF、EGF、PDGF、TGF-β中的任意一种或两种以上的组合。
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Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110027257A1 (en) * | 2008-01-07 | 2011-02-03 | Gwo Rei Biomedical Technology Corporation | Clottable concentrate of platelet growth factors and preparation method thereof |
US20140356893A1 (en) * | 2013-06-04 | 2014-12-04 | Allan Mishra | Compositions and methods for using platelet-rich plasma for drug discovery, cell nuclear reprogramming, proliferation or differentiation |
CN105505855A (zh) * | 2016-02-04 | 2016-04-20 | 关志广 | 一种具备增殖能力的自体表皮细胞apg凝胶的制作方法 |
US20160263193A1 (en) * | 2013-11-14 | 2016-09-15 | Nte-Sener Healthcare, S.A. | Method for obtaining a cytokine-rich composition and composition obtained by means of this method |
CN106692196A (zh) * | 2017-02-09 | 2017-05-24 | 深圳市合康生物科技股份有限公司 | 一种自体美容微针制剂的制备方法及其应用 |
US9757418B1 (en) * | 2009-07-09 | 2017-09-12 | Pgfx Patent Holdings, Llc | Process for removing growth factors from platelets |
CN111690610A (zh) * | 2020-07-22 | 2020-09-22 | 暨赛再生医学科技有限公司 | 一种高效诱导培养制备自然杀伤性nk细胞的方法 |
CN112625084A (zh) * | 2021-01-06 | 2021-04-09 | 广西千度科技发展集团有限公司 | 一种大规模分离干细胞旁分泌蛋白与分泌体囊泡的方法 |
CN113730438A (zh) * | 2021-09-03 | 2021-12-03 | 秦红 | 条件血清以及细胞滤液中富含细胞因子的制备方法 |
CN114146096A (zh) * | 2021-11-25 | 2022-03-08 | 成都清科生物科技有限公司 | 一种富含细胞因子的条件血清的制备方法及应用 |
CN114344257A (zh) * | 2022-01-25 | 2022-04-15 | 广州安立美健康生物科技有限公司 | 一种自体脂肪酸蛋白乳的制备方法以及应用 |
-
2022
- 2022-07-08 CN CN202210807378.7A patent/CN115125202B/zh active Active
Patent Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110027257A1 (en) * | 2008-01-07 | 2011-02-03 | Gwo Rei Biomedical Technology Corporation | Clottable concentrate of platelet growth factors and preparation method thereof |
US9757418B1 (en) * | 2009-07-09 | 2017-09-12 | Pgfx Patent Holdings, Llc | Process for removing growth factors from platelets |
US20140356893A1 (en) * | 2013-06-04 | 2014-12-04 | Allan Mishra | Compositions and methods for using platelet-rich plasma for drug discovery, cell nuclear reprogramming, proliferation or differentiation |
US20160263193A1 (en) * | 2013-11-14 | 2016-09-15 | Nte-Sener Healthcare, S.A. | Method for obtaining a cytokine-rich composition and composition obtained by means of this method |
CN105505855A (zh) * | 2016-02-04 | 2016-04-20 | 关志广 | 一种具备增殖能力的自体表皮细胞apg凝胶的制作方法 |
CN106692196A (zh) * | 2017-02-09 | 2017-05-24 | 深圳市合康生物科技股份有限公司 | 一种自体美容微针制剂的制备方法及其应用 |
CN111690610A (zh) * | 2020-07-22 | 2020-09-22 | 暨赛再生医学科技有限公司 | 一种高效诱导培养制备自然杀伤性nk细胞的方法 |
CN112625084A (zh) * | 2021-01-06 | 2021-04-09 | 广西千度科技发展集团有限公司 | 一种大规模分离干细胞旁分泌蛋白与分泌体囊泡的方法 |
CN113730438A (zh) * | 2021-09-03 | 2021-12-03 | 秦红 | 条件血清以及细胞滤液中富含细胞因子的制备方法 |
CN114146096A (zh) * | 2021-11-25 | 2022-03-08 | 成都清科生物科技有限公司 | 一种富含细胞因子的条件血清的制备方法及应用 |
CN114344257A (zh) * | 2022-01-25 | 2022-04-15 | 广州安立美健康生物科技有限公司 | 一种自体脂肪酸蛋白乳的制备方法以及应用 |
Non-Patent Citations (2)
Title |
---|
王书军等: "不同套装制备的富血小板血浆中细胞及细胞因子成分的比较", 《中华关节外科杂志(电子版)》 * |
陈曦等: "富含细胞因子自体血清的制备及细胞因子分析", 《中国组织工程研究》 * |
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